Modified nucleosides for the treatment of viral infections and abnormal cellular proliferation

ABSTRACT

The disclosed invention is a composition for and a method of treating a Flaviviridae (including BVDV and HCV), Orthomyxoviridae (including Influenza A and B) or Paramyxoviridae (including RSV) infection, or conditions related to abnormal cellular proliferation, in a host, including animals, and especially humans, using a nucleoside of general formula (I)-(XXIII) or its pharmaceutically acceptable salt or prodrug.  
     This invention also provides an effective process to quantify the viral load, and in particular BVDV, HCV or West Nile Virus load, in a host, using real-time polymerase chain reaction (“RT-PCR”). Additionally, the invention discloses probe molecules that can fluoresce proportionally to the amount of virus present in a sample.

[0001] This application claims priority to U.S. provisional applicationNo. 60/241,488, filed Oct. 18, 2000 and U.S. provisional application No.60/282,156, filed on Apr. 6, 2001.

FIELD OF THE INVENTION

[0002] The present invention includes compounds and methods for thetreatment of Flaviviridae, Orthomyxoviridae, Paramyxoviridae infectionsand abnormal cellular proliferation.

BACKGROUND OF THE INVENTION

[0003] Flavirididae

[0004] The Flaviviridae is a group of positive single-stranded RNAviruses with a genome size from 9-15 kb. They are enveloped viruses ofapproximately 40-50 nm. An overview of the Flaviviridae taxonomy isavailable from the International Committee for Taxonomy of Viruses. TheFlaviviridae consists of three genera.

[0005] 1. Flaviviruses. This genus includes the Dengue virus group(Dengue virus, Dengue virus type 1, Dengue virus type 2, Dengue virustype 3, Dengue virus type 4), the Japanese encephalitis virus group(Alfuy Virus, Japanese encephalitis virus, Kookaburra virus, Koutangovirus, Kunjin virus, Murray Valley encephalitis virus, St. Louisencephalitis virus, Stratford virus, Usutu virus, West Nile Virus), theModoc virus group, the Rio Bravo virus group (Apoi virus, Rio Brovovirus, Saboya virus), the Ntaya virus group, the Tick-Borne encephalitisgroup (tick bom encephalitis virus), the Tyuleniy virus group, Uganda Svirus group and the Yellow Fever virus group. Apart from these majorgroups, there are some additional Flaviviruses that are unclassified.

[0006] 2. Hepaciviruses. This genus contains only one species, theHepatitis C virus (HCV), which is composed of many clades, types andsubtypes.

[0007] 3. Pestiviruses. This genus includes Bovine Viral DiarrheaVirus-2 (BVDV-2), Pestivirus type 1 (including BVDV), Pestivirus type 2(including Hog Cholera Virus) and Pestivirus type 3 (including BorderDisease Virus).

[0008] One of the most important Flaviviridae infections in humans iscaused by the hepatitis C virus (HCV). This is the second major cause ofviral hepatitis, with an estimated 170 million carriers world-wide(World Health Organization; Hepatitis C: global prevalence, WeeklyEpidemiological Record, 1997, 72, 341), 3.9 million of whom reside inthe United States (Centers for Disease Control; unpublished data,http://www.cdc.gov/ncidod/diseases/ hepatitis/heptab3.htm).

[0009] The genomic organization of the Flaviviridae share many commonfeatures. The hepatitis C virus (HCV) genome is often used as a model.HCV is a small, enveloped virus with a positive single-stranded RNAgenome of 9.6 kb within the nucleocapsid. The genome contains a singleopen reading frame (ORF) encoding a polyprotein of just over 3,000 aminoacids, which is cleaved to generate the mature structural andnonstructural viral proteins. The ORF is flanked by 5′ and 3′non-translated regions (NTRs) of a few hundred nucleotides in length,which are important for RNA translation and replication. The translatedpolyprotein contains the structural core (C) and envelope proteins (E1,E2, p7) at the N-terminus, followed by the nonstructural proteins (NS2,NS3, NS4A, NS4B, NS5A, NS5B). The mature structural proteins aregenerated via cleavage by the host signal peptidase (see: Hijikata, M.et al. Proc. Nat. Acad. Sci., USA, 1991, 88, 5547; Hussy, P. et al.Virology, 1996, 224, 93; Lin, C. et al. J. Virol., 1994, 68, 5063;Mizushima, H. et al. J. Virol., 1994, 68, 2731; Mizushima, H. et al. J.Virol., 1994, 68, 6215; Santolini, E. et al. J. Virol., 1994, 68, 3631;Selby, M. J. et al. Virology, 1994, 204, 114; and Grakoui, A. et al.Proc. Nat. Acad. Sci., USA, 1993, 90, 10538). The junction between NS2and NS3 is autocatalytically cleaved by the NS2/NS3 protease (see:Hijikata, M. et al. J. Virol., 1993, 67, 4665 and Bartenschlager, R. etal. J. Virol., 1994, 68, 5045), while the remaining four junctions arecleaved by the N-terminal serine protease domain of NS3 complexed withNS4A. (see: Failla, C. et al. J. Virol., 1994, 68, 3753; Lin, C. et al.J. Virol., 1994, 68, 8147; Tanji, Y. et al. J. Virol., 1995, 69, 1575and Tai, C. L. et al. J. Virol., 1996, 70, 8477) The NS3 protein alsocontains the NTP-dependent helicase activity which unwinds duplex RNAduring replication. The NS5B protein possesses RNA-dependent RNApolymerase (RDRP) activity (see: Behrens, S. E. et al. EMBO J., 1996,15, 12; Lohmann, V. et al. J. Virol., 1997, 71, 8416-8428 and Lohmann,V. et al. Virology, 1998, 249, 108), which is essential for viralreplication. (Ferrari, E. et al. J. Virol., 1999, 73, 1649) It isemphasized here that, unlike HBV or HIV, no DNA is involved in thereplication of HCV. Recently in vitro experiments using NS5B, substratespecificity for HCV-RDRP was studied using guanosine 5′-monophosphate(GMP), 5′-diphosphate (GDP), 5′-triphosphate (GTP) and the5′-triphosphate of 2′-deoxy and 2′,3′-dideoxy guanosine (dGTP and ddGTP,respectively). The authors claimed that HCV-RDRP has a strictspecificity for ribonucleoside 5′-triphosphates and requires the 2′- and3′-OH groups. (Lohmann; Virology, 108) Their experiments suggest thatthe presence of 2′- and 3′-substituents would be the prerequisite fornucleoside 5′-triphosphates to interact with HCV-RDRP and to act assubstrates or inhibitors.

[0010] Examples of antiviral agents that have been identified as activeagainst the hepatitis C flavivirus include:

[0011] 1. Interferon and ribavirin (Battaglia, A. M. et al. Ann.Pharmacother. 2000, 34, 487; Berenguer, M. et al. Antivir. Ther. 1998, 3(Suppl. 3), 125);

[0012] 2. Substrate-based NS3 protease inhibitors (Attwood et al. PCT WO98/22496, 1998; Attwood et al. Antiviral Chemistry and Chemotherapy1999, 10, 259, Attwood et al. German Patent Publication DE 19914474;Tung et al. PCT WO 98/17679), including alphaketoamides andhydrazinoureas, and inhibitors that terminate in an electrophile such asa boronic acid or phosphonate (Llinas-Brunet et. al. PCT WO 99/07734);

[0013] 3. Non-substrate-based inhibitors such as2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,Biochemical and Biophysical Research Commnunications, 1997, 238, 643 andSudo K. et al. Antiviral Chemistry and Chemotherapy 1998, 9, 186),including RD3-4082 and RD3-4078, the former substituted on the amidewith a 14 carbon chain and the latter processing a para-phenoxyphenylgroup;

[0014] 4. Thiazolidine derivatives which show relevant inhibition in areverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5Bsubstrate (Sudo K. et al. Antiviral Research 1996, 32, 9), especiallycompound RD-1-6250, possessing a fused cinnamoyl moiety substituted witha long alkyl chain, RD4 6205 and RD4 6193;

[0015] 5. Thiazolidines and benzanilides identified in Kakiuchi N. etal. J. EBS Letters 421, 217 and Takeshita N. et al. AnalyticalBiochemistry 1997, 247, 242;

[0016] 6. A phenanthrenequinone possessing activity against HCV proteasein a SDS-PAGE and autoradiography assay isolated from the fermentationculture broth of Streptomyces sp., Sch 68631 (Chu M. et al. TetrahedronLetters 1996, 37, 7229), and Sch 351633, isolated from the fungusPenicillium griscofuluum, which demonstrates activity in a scintillationproximity assay (Chu M. et al., Bioorganic and Medicinal ChemistryLetters 9, 1949);

[0017] 7. Selective NS3 inhibitors based on the macromolecule elgin c,isolated from leech (Qasim M. A. et al. Biochemistry 1997, 36,1598);

[0018] 8. HCV helicase inhibitors (Diana G. D. et al., U.S. Pat. No.5,633,358 and Diana G. D. et al. PCT WO 97/36554);

[0019] 9. HCV polymerase inhibitors such as nucleotide analogues,gliotoxin (Ferrari R. et al. Journal of Virology 1999, 73, 1649), andthe natural product cerulenin (Lohmann V. et al. Virology 1998, 249,108);

[0020] 10. Antisense phosphorothioate oligodeoxynucleotides (S—ODN)complementary to at least a portion of a sequence of the HCV (Andersonet al. U.S. Pat. No. 6,174,868), and in particular the sequencestretches in the 5′ non-coding region (NCR) (Alt M. et al. Hepatology1995, 22, 707), or nucleotides 326-348 comprising the 3′ end of the NCRand nucleotides 371-388 located in the core coding region of the HCV RNA(Alt M. et al. Archives of Virology 1997, 142, 589 and Galderisi U. etal., Journal of Cellular Physiology 1999, 81:2151);

[0021] 11. Inhibitors of IRES-dependent translation (Ikeda N et al.Japanese Patent Pub. JP-08268890; Kai Y. et al. Japanese PatentPublication JP-10101591);

[0022] 12. Nuclease-resistant ribozymes (Maccjak D. J. et al.,Hepatology 1999, 30, abstract 995);

[0023] 13. Amantadine, such as rimantadine (Smith, Abstract from AnnualMeeting of the American Gastoenterological Association and AASLD, 1996);

[0024] 14. Quinolones, such as ofloxacin, ciprofloxacin and levofloxacin(AASLD Abstracts, Hepatology, October 1994, Program Issue, 20 (4), pt.2,abstract no. 293);

[0025] 15. Nucleoside analogs (Ismaili et al. WO 01/60315; Storer WO01/32153), including 2′-deoxy-L-nucleosides (Watanabe et al. WO01/34618), and 1-(β-L-ribofuranosyl)-1,2,4-triazole-3-carboxamide(levovirinTM) (Tam WO 01/46212); and

[0026] 16. Other miscellaneous compounds including1-amino-alkylcyclohexanes (Gold et al. U.S. Pat. No. 6,034,134), alkyllipids (Chojkier et al. U.S. Pat. No. 5,922,757), vitamin E and otherantioxidants (Chojkier et al. U.S. Pat. No. 5,922,757), squalene, bileacids (Ozeki et al. U.S. Pat. No. 5,846,964),N-(phosphonoacetyl)-L-aspartic acid, (Diana et al. U.S. Pat. No.5,830,905), benzenedicarboxamides (Diana et al. U.S. Pat. No.5,633,388), polyadenylic acid derivatives (Wang et al. U.S. Pat. No.5,496,546), 2′,3′-dideoxyinosine (Yarchoan et al. U.S. Pat. No.5,026,687), benzimidazoles (Colacino et al. U.S. Pat. No. 5,891,874),glucamines (Mueller et al. WO 01/08672),substituted-1,5-imino-D-glucitol compounds (Mueller et al. WO 00/47198).

[0027] Orthomyxoviridae

[0028] The Orthomyxoviridae is a group of segmented negativesingle-stranded RNA viruses with a genome size from 10-13.6 kb. They areenveloped viruses of approximately 80-120 nm. An overview of theOrthomyxoviridae taxonomy is available from the International Committeefor Taxonomy of Viruses. The Orthomyxoviridae consists of three genera,which can be distinguished on the basis of antigenic differences betweentheir nucleocapsid (NP) and matrix proteins (M).

[0029] 1. Influenzavirus A, B. This genus contains influenza A and Bviruses each of which contain eight distinct RNA segments. Influenza Bviruses show little variability in their surface glycoproteins and onlyinfect humans. On the other hand, influenza A viruses have greatvariability in their surface glycoproteins of influenza A viruses, andthey can be divided into subtypes based on the antigenic nature of theirhemagglutinin (HA) and neuroamidase (NA) glycoproteins and infect humansas well as swine, horses, seals, fowl, ducks and many other species ofbirds.

[0030] 2. Influenzavirus C. This genus contains only one species,influenza C, which contains only seven distinct RNA segments. InfluenzaC only has a single multifunctional glycoprotein and infects mainlyhumans, but has also been isolated from swine in China.

[0031] 3. Influenzavirus D. This genus contains influenza D, which issolely tick-borne viruses that are structurally and genetically similarto influenza A, B and C.

[0032] One of the most important Orthomyxoviridae infections in humansis caused by the influenza A virus. These viruses are highly contagiousand cause acute respiratory illness that has plagued society in epidemicproportions since ancient times. One of the earliest recordings of aninfluenza A epidemic can be traced to Hippocrates in 412 BC. Theseepidemics are rather frequent and are often fatal to the elderly,however these epidemics are quite unpredictable. These viruses areunique respiratory tract viruses, in that they undergo significantantigenic variation. Both hemagglutinin (HA) and neuroamidase (NA)glycoproteins are capable of antigenic drifts and shifts. There arefourteen known hemagglutinin (H1-H14) glycoproteins and nine knownneuroamidase (N1-N9) glycoproteins. For example, since the first humaninfluenza virus was isolated in 1933, major antigenic shifts haveoccurred. In 1957, the H2N2 subtype (Asian influenza) replaced the H1N1subtype (Spanish influenza). Currently, the primary subtypes ofinfluenza are H1N1, which reappeared in 1977 and H3N2, which reappearedin 1968.

[0033] The vast majority of research on influenza virus gene expressionand RNA replication has been carried out with the influenza A virus. Themost striking feature of the influenza A virion is a layer of about 500spikes radiating outward (10 to 14 nm) from the lipid envelope. Thesespikes are of two types: rod-shaped spikes of HA and mushroom-shapedspikes of NA. The ratio of HA and NA varies, but is usually 4-5 to 1.Each gene segment encodes its own proteins, with the exception of theseventh and eighth, which encodes M₁ and M₂, and NS₁ and NS₂respectively. The first 12 nucleotides at the 3′-end and the first 13nucleotides at the 5′-end of each vRNA segment are conserved in alleight RNA segments. The first gene to have its nucleotide sequencedetermined was HA. Since then, all 14 known HA antigenic subtpes andmany variants within the subtypes have been determined.

[0034] In infected cells, the vRNAs are both transcribed into mRNAs andreplicated. The synthesis of mRNA is distinct, in that the RNA is primedby 5′ capped fragments derived from newly synthesized host-cell RNApolymerase II transcripts. The mRNA chain elongates until a stretch ofuridine residues is reached 15-22 nucleotides before the 5′-ends of thevRNAs where transcription ends and polyadenylate is added to the mRNAs.For replication to occur, an alternative type of transcription isrequired that results in the production of full-length copies of thevRNAs. The full-length transcripts are initiated without a primer andare not terminated at the poly(A) site used during mRNA synthesis. Thesecond step in replication is the copying of the template RNAs intovRNAs. This synthesis also occurs without a primer, since the vRNAscontain 5′-triphosphorylated ends. All three types of virus-specificRNAs—mRNA, template RNA and vRNA—are synthesized in the nucleus.

[0035] Examples of antiviral agents that have been identified as activeagainst the influenza A virus include:

[0036] 1. Actinomycin D (Barry, R. D. et al. “Participation ofdeoxyribonucleic acid in the multiplication of influenza virus” Nature,1962, 194, 1139-1140);

[0037] 2. Amantadine (Van Voris, L. P. et al. “Antivirals for thechemoprophylaxis and treatment of influenza” Semin Respir Infect, 1992,7, 61-70);

[0038] 3,4-Amino- or4-guanidino-2-deoxy-2,3-didehydro-D-N-acetylneuroaminic acid -4-amino-or 4-guanidino-Neu 5 Ac2en (von Itzstein, M. et al. “Rational design ofpotent sialidase-based inhibitors of influenza virus replication”Nature, 1993, 363, 418-423);

[0039] 4. Ribavirin (Van Voris, L. P. et al. “Antivirals for thechemoprophylaxis and treatment of influenza” Semin Respir Infect, 1992,7, 61-70);

[0040] 5. Interferon (Came, P. E. et al. “Antiviral activity of aninterferon-inducing synthetic polymer” Proc Soc Exp Biol Med, 1969, 131,443-446; Gerone, P. J. et al. “Inhibition of respiratory virusinfections of mice with aeresols of synthetic double-strandedribonucleic acid” Infect Immun, 1971, 3, 323-327; Takano, K. et al.“Passive interferon protection in mouse influenza” J Infect Dis, 1991,164, 969-972);

[0041] 6. Inactivated influenza A and B virus vaccines (“Clinicalstudies on influenza vaccine—1978” Rev Infect Dis, 1983, 5, 721-764;Galasso, G. T. et al. “Clinical studies on influenza vaccine—1976” JInfect Dis, 1977, 136 (suppl), S341-S746; Jennings, R. et al. “Responsesof volunteers to inactivated influenza virus vaccines” J Hyg, 1981, 86,1-16; Kilbourne, E. D. “Inactivated influenza vaccine” In: Plothin S A,Mortimer EA, eds. Vaccines Philadelphia: Saunders, 1988, 420-434; Meyer,H. M., Jr. et al. “Review of existion vaccines for influenza” Am J ClinPathol, 1978, 70, 146-152; “Mortality and Morbidity Weekly Report.Prevention and control of Influenza: Part I, Vaccines. Recommendationsof the Advisory Committee on Immunication Practices (ACIP)” MMWR, 1993,42 (RR-6), 1-14; Palache, A. M. et al. “Antibody response afterinfluenza immunization with various vaccine doses: A double-blind,placebo-controlled, multi-centre, dose-response study in elderlynursing-home residents and young volunteers” Vaccine, 1993, 11,3-9;Potter, C. W. “Inactivated influenza virus vaccine” In: Beare AS, ed.Basic and applied influeza research, Boca Raton, Fla.: CRC Press, 1982,119-158).

[0042] Paramyxoviridae

[0043] The Paramyxoviridae is a group of negative single-stranded RNAviruses with a genome size from 16-20 kb. They are enveloped viruses ofapproximately 150-300 nm. An overview of the Paramyxoviridae taxonomy isavailable from the International Committee for Taxonomy of Viruses. TheParamyxoviridae consists of two subfamilies.

[0044] 1. Paramyxovirinae. This subfamily contains three genera:

[0045] a) Paramyxovirus. This genus is represented by Sendai virus andincluding human parainfluenza viruses 1 and 3;

[0046] b) Rubulavirus. This genus is represented by the mumps virus,simian virus 5, Newcastle disease virus and the human parainfluenzaviruses 2 and 4;

[0047] c) Morbillivirus. This genus is represented by the measles virus;and

[0048] 2. Pneumovirinae. This subfamily encode a larger number of mRNAsthan the other sub-family (ten, compared with six or seven) and containsonly one genera:

[0049] a) Pneumovirus. This genus is best represented by the respiratorysyncytial virus (RSV), but also includes bovine (BRSV), ovine RSV(ORSC), caprine RSV (CRSV), pneumonia virus of mice (PVM) and turkeyrhinotracheitis virus (TRTV).

[0050] One of the most important Pneumovirinae infections in humans iscaused by the respiratory syncytial virus (RSV). RSV is the mostimportant cause of viral lower respiratory tract disease in infants andchildren worldwide. In most areas, RSV outranks all other microbialpathogens as a cause of pneumonia and bronchiolitis in infants under oneyear of age. It has also been found that RSV infection is an importantagent of disease in immunosuppressed adults and in the elderly.Additionally, BRSV has been shown to be an economically importantdisease in cattle.

[0051] The 3′-end of genomic RSV RNA consists of a 44-nucleotideextragenic leader region that is presumed to contain the major viralpromoter. The leader region is followed by the ten viral genes, which isfollowed by a 155-nucleotide extragenic trailer region. Eighty eightpercent of the genomic RNA is accounted for by the ORFs for the tenmajor proteins. Each gene begins with a conserved nine-nucleotidegene-start signal. For each gene, transcription begins at the firstnucleotide of the signal. Each gene terminates with a semi-conserved 12to 13 nucleotide gene-end signal that directs transcriptionaltermination and polyadenylation. The first nine genes arenon-overlapping and are separated by intergenic regions that range insize from 1 to 52 nucleotides. The intergenic regions do not contain anyconserved sequence motifs or any obvious features of secondarystructure. The last two RSV genes overlap by 68 nucleotides. Thus, oneof the gene-start signals is located inside of, rather than after theother gene.

[0052] Examples of antiviral agents that have been identified as activeagainst RSV include:

[0053] 1. Ribavirin (Hruska, J. F. et al. “In vivo inhibition ofrespiratory syncytial virus by ribavirin” Antimicrob Agents Chemother,1982, 21, 125-130); and

[0054] 2. Purified human intravenous IgG—IVIG (Prince, G. A. et al.“Effectiveness of topically administered neutralizing antibodies inexperimental immunotherapy of respiratory syncytial virus infection incotton rats” J Virol, 1987, 61, 1851-1954; Prince, G. A. et al.“Immunoprophylaxis and immunotherapy of respiratory syncytial virusinfection in cotton rats” Infect Immun, 1982, 42, 81-87).

[0055] Abnormal Cellular Proliferation

[0056] Cellular differentiation, growth, function and death areregulated by a complex network of mechanisms at the molecular level in amulticellular organism. In the healthy animal or human, these mechanismsallow the cell to carry out its designed function and then die at aprogrammed rate.

[0057] Abnormal cellular proliferation, notably hyperproliferation, canoccur as a result of a wide variety of factors, including geneticmutation, infection, exposure to toxins, autoimmune disorders, andbenign or malignant tumor induction.

[0058] There are a number of skin disorders associated with cellularhyperproliferation. Psoriasis, for example, is a benign disease of humanskin generally characterized by plaques covered by thickened scales. Thedisease is caused by increased proliferation of epidermal cells ofunknown cause. In normal skin the time required for a cell to move fromthe basal layer to the upper granular layer is about five weeks. Inpsoriasis, this time is only 6 to 9 days, partially due to an increasein the number of proliferating cells and an increase in the proportionof cells which are dividing (G. Grove, Int. J. Dermatol. 18:111, 1979).Approximately 2% of the population in the United States have psoriasis,occurring in about 3% of Caucasian Americans, in about 1% of AfricanAmericans, and rarely in native Americans. Chronic eczema is alsoassociated with significant hyperproliferation of the epidermis. Otherdiseases caused by hyperproliferation of skin cells include atopicdermatitis, lichen planus, warts, pemphigus vulgaris, actinic keratosis,basal cell carcinoma and squamous cell carcinoma.

[0059] Other hyperproliferative cell disorders include blood vesselproliferation disorders, fibrotic disorders, autoimmune disorders,graft-versus-host rejection, tumors and cancers.

[0060] Blood vessel proliferative disorders include angiogenic andvasculogenic disorders. Proliferation of smooth muscle cells in thecourse of development of plaques in vascular tissue cause, for example,restenosis, retinopathies and atherosclerosis. The advanced lesions ofatherosclerosis result from an excessive inflammatory-proliferativeresponse to an insult to the endothelium and smooth muscle of the arterywall (Ross, R. Nature, 1993, 362:801-809). Both cell migration and cellproliferation play a role in the formation of atherosclerotic lesions.

[0061] Fibrotic disorders are often due to the abnormal formation of anextracellular matrix. Examples of fibrotic disorders include hepaticcirrhosis and mesangial proliferative cell disorders. Hepatic cirrhosisis characterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. Hepatic cirrhosis cancause diseases such as cirrhosis of the liver. An increasedextracellular matrix resulting in a hepatic scar can also be caused byviral infection such as hepatitis. Lipocytes appear to play a major rolein hepatic cirrhosis.

[0062] Mesangial disorders are brought about by abnormal proliferationof mesangial cells. Mesangial hyperproliferative cell disorders includevarious human renal diseases, such as glomerulonephritis, diabeticnephropathy, malignant nephrosclerosis, thrombotic micro-angiopathysyndromes, transplant rejection, and glomerulopathies.

[0063] Another disease with a proliferative component is rheumatoidarthritis. Rheumatoid arthritis is generally considered an autoimmunedisease that is thought to be associated with activity of autoreactive Tcells (See, e.g., Harris, E. D., Jr., The New England Journal ofMedicine, 1990, 322: 1277-1289), and to be caused by autoantibodiesproduced against collagen and IgE.

[0064] Other disorders that can include an abnormal cellularproliferative component include Behcet's syndrome, acute respiratorydistress syndrome (ARDS), ischemic heart disease, post-dialysissyndrome, leukemia, acquired immune deficiency syndrome, vasculitis,lipid histiocytosis, septic shock and inflammation in general.

[0065] A tumor, also called a neoplasm, is a new growth of tissue inwhich the multiplication of cells is uncontrolled and progressive. Abenign tumor is one that lacks the properties of invasion and metastasisand is usually surrounded by a fibrous capsule. A malignant tumor (i.e.,cancer) is one that is capable of both invasion and metastasis.Malignant tumors also show a greater degree of anaplasia (i.e., loss ofdifferentiation of cells and of their orientation to one another and totheir axial framework) than benign tumors.

[0066] Approximately 1.2 million Americans are diagnosed with cancereach year, 8,000 of which are children. In addition, 500,000 Americansdie from cancer each year in the United States alone. Prostate and lungcancers are the leading causes of death in men while breast and lungcancer are the leading causes of death in women. It is estimated thatcancer-related costs account for about 10 percent of the total amountspent on disease treatment in the United States(CNN.Cancer.Factshttp://www.cnn.com/HEALTH/9511/conquer_cancer/facts/index.html,page 2 of 2, Jul. 18, 1999).

[0067] Proliferative disorders are currently treated by a variety ofclasses of compounds including alkylating agents, antimetabolites,natural products, enzymes, biological response modifiers, miscellaneousagents, radiophannaceuticals (for example, Y-90 tagged to hormones orantibodies), hormones and antagonists, such as those listed below.

[0068] Alkylating Agents

[0069] Nitrogen Mustards: Mechlorethamine (Hodgkin's disease,non-Hodgkin's lymphomas), Cyclophosphamide, Ifosfamide (acute andchronic lymphocytic leukemias, Hodgkin's disease, non-Hodgkin'slymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung, Wilms'tumor, cervix, testis, soft-tissue sarcomas), Melphalan (L-sarcolysin)(multiple myeloma, breast, ovary), Chlorambucil (chronic lymphocticleukemia, primary macroglobulinemia, Hodgkin's disease, non-Hodgkin'slymphomas).

[0070] Ethylenimines and Methylmelamines: Hexamethylmelamine (ovary),Thiotepa (bladder, breast, ovary).

[0071] Alkyl Sulfonates: Busulfan (chronic granuloytic leukemia).

[0072] Nitrosoureas: Carmustine (BCNU) (Hodgkin's disease, non-Hodgkin'slymphomas, primary brain tumors, multiple myeloma, malignant melanoma),Lomustine (CCNU) (Hodgkin's disease, non-Hodgkin's lymphomas, primarybrain tumors, small-cell lung), Semustine (methyl-CCNU) (primary braintumors, stomach, colon), Streptozocin (STR) (malignant pancreaticinsulinoma, malignant carcinoin).

[0073] Triazenes: Dacarbazine (DTIC;dimethyltriazenoimidazole-carboxamide) (malignant melanoma, Hodgkin'sdisease, soft-tissue sarcomas).

[0074] Antimetabolites

[0075] Folic Acid Analogs: Methotrexate (amethopterin) (acutelymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast, headand neck, lung, osteogenic sarcoma).

[0076] Pyrimidine Analogs: Fluorouracil (5-fluorouracil; 5-FU)Floxuridine (fluorodeoxyuridine; FUdR) (breast, colon, stomach,pancreas, ovary, head and neck, urinary bladder, premalignant skinlesions) (topical), Cytarabine (cytosine arabinoside) (acutegranulocytic and acute lymphocytic leukemias).

[0077] Purine Analogs and Related Inhibitors: Mercaptopurine(6-mercaptopurine; 6-MP) (acute lymphocytic, acute granulocytic andchronic granulocytic leukemia), Thioguanine (6-thioguanine: TG) (acutegranulocytic, acute lymphocytic and chronic granulocytic leukemia),Pentostatin (2′-deoxycyoformycin) (hairy cell leukemia, mycosisflingoides, chronic lymphocytic leukemia).

[0078] Vinca Alkaloids: Vinblastine (VLB) (Hodgkin's disease,non-Hodgkin's lymphomas, breast, testis), Vincristine (acute lymphocyticleukemia, neuroblastoma, Wilms' tumor, rhabdomyosarcoma, Hodgkin'sdisease, non-Hodgkin's lymphomas, small-cell lung).

[0079] Epipodophylotoxins: Etoposide (testis, small-cell lung and otherlung, breast, Hodgkin's disease, non-Hodgkin's lymphomas, acutegranulocytic leukemia, Kaposi's sarcoma), Teniposide (testis, small-celllung and other lung, breast, Hodgkin's disease, non-Hodgkin's lymphomas,acute granulocytic leukemia, Kaposi's sarcoma).

[0080] Natural Products

[0081] Antibiotics: Dactinomycin (actinonmycin D) (choriocarcinoma,Wilms' tumor rhabdomyosarcoma, testis, Kaposi's sarcoma), Daunorubicin(daunomycin; rubidomycin) (acute granulocytic and acute lymphocyticleukemias), Doxorubicin (soft tissue, osteogenic, and other sarcomas;Hodgkin's disease, non-Hodgkin's lymphomas, acute leukemias, breast,genitourinary thyroid, lung, stomach, neuroblastoma), Bleomycin (testis,head and neck, skin and esophagus lung, and genitourinary tract,Hodgkin's disease, non-Hodgkin's lymphomas), Plicamycin (mithramycin)(testis, malignant hypercalcema), Mitomycin (mitomycin C) (stomach,cervix, colon, breast, pancreas, bladder, head and neck).

[0082] Enzymes: L-Asparaginase (acute lymphocytic leukemia).

[0083] Biological Response Modifiers: Interferon-alfa (hairy cellleukemia, Kaposi's sarcoma, melanoma, carcinoid, renal cell, ovary,bladder, non Hodgkin's lymphomas, mycosis fungoides, multiple myeloma,chronic granulocytic leukemia).

[0084] Miscellaneous Agents

[0085] Platinum Coordination Complexes: Cisplatin (cis-DDP) Carboplatin(testis, ovary, bladder, head and neck, lung, thyroid, cervix,endometrium, neuroblastoma, osteogenic sarcoma).

[0086] Anthracenedione: Mixtozantrone (acute granulocytic leukemia,breast).

[0087] Substituted Urea: Hydroxyurea (chronic granulocytic leukemia,polycythemia vera, essential thrombocytosis, malignant melanoma).

[0088] Methylhydrazine Derivative: Procarbazine (N-methylhydrazine, M1H)(Hodgkin's disease).

[0089] Adrenocortical Suppressant: Mitotane (o,p′-DDD) (adrenal cortex),Amino-glutethimide (breast).

[0090] Adrenorticosteriods: Prednisone (acute and chronic lymphocyticleukemias, non-Hodgkin's lymphomas, Hodgkin's disease, breast).

[0091] Progestins: Hydroxprogesterone caproate, Medroxyprogesteroneacetate, Megestrol acetate (endometrium, breast). Anti-angiogenesisAgents Angiostatin, Endostatin.

[0092] Hormones and Antagonists

[0093] Estrogens: Diethylstibestrol Ethinyl estradiol (breast, prostate)

[0094] Antiestrogen: Tamoxifen (breast).

[0095] Androgens: Testosterone propionate Fluxomyesterone (breast).

[0096] Antiandrogen: Flutamide (prostate).

[0097] Gonadotropin-Releasing Hormone Analog: Leuprolide (prostate).

[0098] Toxicity associated with therapy for abnormally proliferatingcells, including cancer, is due in part to a lack of selectivity of thedrug for diseased versus normal cells. To overcome this limitation,therapeutic strategies that increase the specificity and thus reduce thetoxicity of drugs for the treatment of proliferative disorders are beingexplored. One such strategy that is being aggressively pursued is drugtargeting.

[0099] In view of the severity of these diseases and their pervasivenessin animals, including humans, it is an object of the present inventionto provide a compound, method and composition for the treatment of ahost, including animals and especially humans, infected with any of theviruses described above, including flavivirus or pestivirus, influenzavirus or Respiratory Syncytial Virus (“RSV”).

[0100] It is another object of the present invention to provide a methodand composition for the treatment of a host, including animals andespecially humans, with abnormal cellular proliferation.

[0101] It is a further object to provide a method and composition forthe treatment of a host, including animals and especially humans,infected with hepatitis C or BVDV.

[0102] It is a further object to provide a method and composition forthe treatment of a host, including animals and especially humans,infected with influenza.

[0103] It is a further object to provide a method and composition forthe treatment of a host, including animals and especially humans,infected with RSV.

[0104] It is a further object to provide a method and composition forthe treatment of a host, including animals and especially humans, with atumor, including a malignant tumor.

[0105] It is yet another object of the present invention to provide amore effective process to quantify viral load, and in particular of BVDVor HCV load, in a host, including animals, especially humans.

SUMMARY OF THE INVENTION

[0106] The present invention provides a β-D or β-L nucleoside of formula(I)-(XXIII) or its pharmaceutically acceptable salt or prodrug for thetreatment of a host infected with a virus belonging to the Flaviviridae,Orthomyxoviridae and Paramyxoviridae family. Alternatively, the β-D orβ-L nucleoside (I)-(XXIII) or its pharmaceutically acceptable salt orprodrug can be used for the treatment of abnormal cellularproliferation.

[0107] Specifically, the invention also includes methods for treating orpreventing the following:

[0108] (a) a Flaviviridae infection, including all members of theHepacivirus genus (HCV), Pestivirus genus (BVDV, CSFV, BDV), orFlavivirus genus (Dengue virus, Japanese encephalitis virus group(including West Nile Virus), and Yellow Fever virus);

[0109] (b) an Orthomyxoviridae infection, including all members of theInfluenza A, B genus, in particular influenza A and all relevantsubtypes—including H1N1 and H3N2—and Influenza B;

[0110] (c) a Paramyxoviridae infection including Respiratory SyncytialVirus (RSV) infection; and

[0111] (d) abnormal cellular proliferation, including malignant tumors.

[0112] In one embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a β-D nucleoside of the general formula (I) or(II):

[0113] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0114] each D is hydrogen, alkyl, acyl, monophosphate, diphosphate,triphosphate, monophosphate ester, diphosphate ester, triphosphateester, phospholipid or amino acid;

[0115] each W¹ and W² is independently CH or N;

[0116] each X¹ and X² is independently hydrogen, halogen (F, Cl, Br orI), NH₂, NHR⁴, NR⁴R^(4′), NHOR⁴, NR⁴N^(4′)R^(4″), OH, OR⁴, SH or SR⁴;

[0117] each Y¹ is O, S or Se;

[0118] each Z is CH₂ or NH;

[0119] each R¹ and R^(1′) is independently hydrogen, lower alkyl, loweralkenyl, lower alkynyl, aryl, alkylaryl, halogen (F, Cl, Br or I), NH₂,NHR⁵, NR⁵R^(5′), NHOR⁵, NR⁵NHR^(5′), NR⁵NR^(5′)R^(5″), OH, OR⁵, SH, SR⁵,NO₂, NO, CH₂OH, CH₂OR⁵, CO₂H, CO₂R⁵, CONH₂, CONHR⁵, CONR⁵R^(5′) or CN;

[0120] each R² and R^(2′) independently is hydrogen or halogen (F, Cl,Br or I), OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, CH═CH₂, CN, CH₂NH₂, CH₂OH,CO₂H.

[0121] each R³ and R^(3′) independently is hydrogen or halogen (F, Cl,Br or I), OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, CH₃, C₂H₅, CH═CH₂, CN, CH₂NH₂,CH₂OH, CO₂H.

[0122] each R⁴, R^(4′), R^(4″), R⁵, R^(5′) and R⁵″ independently ishydrogen, lower alkyl, lower alkenyl, aryl, or arylalkyl such asunsubstituted or substituted phenyl or benzyl;

[0123] such that for each nucleoside of the general formula (1) or (II),at least one of R² and R^(2′) is hydrogen and at least one of R³ andR^(3′) is hydrogen.

[0124] In another embodiment of the invention, anti-virally oranti-proliferatively effective nucleoside is a β-L nucleoside of thegeneral formula (III) or (IV):

[0125] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0126] each D, W¹, W² X¹, X², Y¹, Z, R¹, R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously;

[0127] such that for each nucleoside of the general formula (III) or(IV), at least one of R² and R^(2′) is hydrogen and at least one of R³and R^(3′) is hydrogen.

[0128] In one embodiment of the invention, the anti-virally oranti-proliferatively effective nucleoside is a β-D-carba-sugarnucleoside of the general formula (V) to (VII):

[0129] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0130] D, W¹, W², X¹, X², Y¹, Z, R¹, R^(1′), R², R^(2′), R³ and R^(3′)is the same as defined previously;

[0131] such that for each nucleoside of the general formula (V) or (VI),at least one of R² and R^(2′) is hydrogen and at least one of R³ andR^(3′) is hydrogen.

[0132] In one embodiment, anti-virally or anti-proliferatively effectivenucleoside is a β-L-carba-sugar nucleoside of the general formula (VIII)to (X):

[0133] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0134] each D, W¹, W², X¹, X², Y¹, Z, R¹, R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously;

[0135] such that for each nucleoside of the general formula (VIII) or(IX), at least one of R² and R² is hydrogen and at least one of R³ andR^(3′) is hydrogen.

[0136] In further embodiment of the invention, the anti-virally oranti-proliferatively effective β-D or β-L-nucleoside is of the generalformula (XI) or (XII), respectively:

[0137] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0138] each D, W¹, W², X¹, X², Y¹, Z, R¹, R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously;

[0139] each Z¹ and Z² independently is O, S, CH₂, NR⁶ or Se;

[0140] each R⁶ is hydrogen, lower alkyl or lower acyl.

[0141] In a further embodiment of this invention, the anti-virally oranti-proliferatively effective ED or β-L-nucleoside, though preferablyβ-D, is of the general formula (XIII):

[0142] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0143] each D, R¹, R^(1′), R², R^(2′), R³ and R^(3′) is the same asdefined previously;

[0144] each Y² is O, S, NH or NR⁷;

[0145] each Y³ is O, S, NH or NR⁸;

[0146] each X³ is OR⁹ or SR⁹; and

[0147] each R⁷, R⁸ and R⁹ is hydrogen, lower alkyl of C₁-C₆, arylalkylor aryl;

[0148] such that for each nucleoside of the general formula (XIII-d), atleast one of R² and R^(2′) is hydrogen and at least one of R³ and R^(3′)is hydrogen.

[0149] In another embodiment, the anti-virally or anti-proliferativelyeffective compound is a β-D or β-L-nucleoside, though preferably β-D,resulting from the addition of a small molecule, such as alkylhypochlorite, alkyl hypobromite, hypobromous acid or acyl halide to anappropriate pyrimidine nucleoside, forming a nucleoside of the formula(XIV):

[0150] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0151] each D, X¹, Y¹, Z, R¹, R², R^(2′), R³ and R^(3′) is the same asdefined previously;

[0152] each L¹ is hydrogen, Cl or Br;

[0153] each L² is OH, OCH₃, OC₂H₅, OC₃H₇, OCF₃, OAc or OBz;

[0154] each Z³ can be O or CH₂.

[0155] In another embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a dimeric nucleoside (each nucleoside being ineither the β-D or β-L configuration) of general formula (XV), in whichthe two nucleosides are linked through a disulfide bond:

[0156] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0157] each D, W¹, W², X¹, Y¹, Z³, R¹, R^(1′), R², R^(2′), R³ and R^(3′)is the same as defined previously.

[0158] In one embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a β-or β-L C-nucleoside of the general formula(XVI):

[0159] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0160] each D, W¹, X¹, X², Y¹, Z, R¹, R², R^(2′), R³ and R^(3′) is thesame as defined previously;

[0161] each W³ is independently N, CH or CR¹;

[0162] each W⁴ and W⁵ is independently N, CH, CX¹ or CR^(1′); and

[0163] each Z⁴ and Z⁵ is independently NH or C(═Y¹);

[0164] such that if Z⁴ and Z⁵ are covalently bound, then Z⁴ is notC(═Y¹) when Z⁵ is C(═Y¹); and

[0165] there are no more than three ring nitrogens.

[0166] In one embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a β-D or β-L-branched-chain sugar nucleoside ofthe general formula (XVII):

[0167] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0168] each D, W¹, W², X¹, X², Z³, R¹, R², R^(2′), R³ and R^(3′) is thesame as defined previously;

[0169] each X⁴ and X⁵ is independently hydrogen, halogen (F, Cl, Br orI), N₃, NH₂, NHR⁸, NR⁸R^(8′), OH, OR⁸, SH or SR⁸; and

[0170] each R⁸ and R^(8′) is independently hydrogen, lower alkyl, loweralkenyl, aryl or arylalkyl,

[0171] such as an unsubstituted or substituted phenyl or benzyl; suchthat for each nucleoside of the general formula (XVII-a) or (XVII-b), X⁴is not OH or OR⁸.

[0172] In one embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a α-D or α-L-nucleoside of the general formula(XVIII):

[0173] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0174] each D, W¹, W², X¹, X², Y¹, R¹, R^(1′), R², R^(2″) R³ and R^(3′)is the same as defined previously;

[0175] In a sub-embodiment of the present invention, the anti-virally oranti-proliferatively effective E-D or β-L nucleoside is of the formula(XIX):

[0176] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0177] each D, R¹, R⁴ and R^(4′) is the same as defined previously;

[0178] each R⁹ is hydrogen, halogen (F, Cl, Br or I) or OP³;

[0179] each P¹ is hydrogen, lower alkyl, lower alkenyl, aryl, arylalkyl(such as an unsubstituted or substituted phenyl or benzyl), OH, OR⁴,NH₂, NHR⁴ or NR⁴R⁴; and

[0180] each P² and P³ is independently hydrogen, alkyl, acyl, -Ms, -Ts,monophosphate, diphosphate, triphosphate, mono-phosphate ester,diphosphate ester, triphosphate ester, phospholipid or amino acid,though preferably hydrogen.

[0181] In a particular sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside ofthe formula (XIX) is the following:

[0182] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0183] each D and P² is the same as defined previously. In a preferredembodiment, D and P² are independently hydrogen.

[0184] In another sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside isof the formula (XX):

[0185] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0186] each D, P¹, P², P³, R¹, R⁴, R^(4′) and R⁹ is the same as definedpreviously.

[0187] In another sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside isof the formula (XXI):

[0188] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0189] each D, P¹, P², P³, R¹, R⁴ and R^(4′) is the same as definedpreviously.

[0190] In a particular sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside ofthe formula (XXI) is the following:

[0191] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0192] each D, P² and P³ is the same as defined previously. In apreferred embodiment, D, P² and P³ are independently hydrogen.

[0193] In another sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside isof the formula (XXII):

[0194] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0195] each D, P¹ and R¹ is the same as defined previously. In apreferred embodiment, D and P² are independently hydrogen.

[0196] In a particular sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside,though preferably β-L, of the formula (XXII) is the following:

[0197] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0198] D is the same as defined previously, and preferably H.

[0199] In another sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside isof the formula (XXIII):

[0200] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0201] each D, P¹, P², P³, R¹, R⁴ and R^(4′) is the same as definedpreviously.

[0202] In a particular sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside ofthe formula (XXIII) is the following:

[0203] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0204] each D, P² and P³ is the same as defined previously. In apreferred embodiment, D, P² and P³ are independently hydrogen.

[0205] In one embodiment, the nucleoside has an EC₅₀ (effectiveconcentration to achieve 50% viral inhibition) when tested in anappropriate cell-based assay, of less than 15 micromolar, and moreparticularly, less than 10 or 5 micromolar. In a preferred embodiment,the nucleoside is enantiomerically enriched.

[0206] The present invention also includes at least the followingfeatures:

[0207] (a) use of a β-D nucleoside or β-L nucleoside of formula(I)-(XXIII), as described herein, or its pharmaceutically acceptablesalt or prodrug thereof in a medical therapy, i.e. as an antiviral orantitumor/anticancer agent, for example for the treatment or prophylaxisof a Flaviviridae infections, including hepatitis C infection;

[0208] (b) use of a β-D nucleoside or β-L nucleoside of formula(I)-(XXIII), as described herein, or its pharmaceutically acceptablesalt or prodrug thereof in the manufacture of a medicament for treatmentof a Flaviviridae infection, including hepatitis C infection;

[0209] (c) a pharmaceutical composition that include an antivirallyeffective amount of a β-D nucleoside or β-L nucleoside of formula(I)-(XXIII), as described herein, or its pharmaceutically acceptablesalt or prodrug thereof together with a pharmaceutically acceptablecarrier or diluent according to the present invention;

[0210] (d) a pharmaceutical composition with a β-D nucleoside or β-Lnucleoside of formula (I)-(XXIII), as described herein, or itspharmaceutically acceptable salt or prodrug thereof in combination withone or more other antivirally effective agents; and

[0211] (e) process for the preparation of β-D nucleoside or β-Lnucleoside of formula (I)-(XXIII), as described herein, and theirpharmaceutically acceptable salts and prodrugs thereof.

[0212] The activity and toxicity of the compounds described herein canbe evaluated according to any known procedure. An efficient process toquantify the viral load in a host using real-time polymerase chainreaction (“RT-PCR”) is provided below. The process involves the use of aquenched fluorescent probe molecule, which can be hybridized to thetarget viral DNA or RNA. Upon exonucleolytic degradation, a detectablefluorescent signal can be monitored. Using this technique, the RT-PCRamplified DNA or RNA can be detected in real time by monitoring thepresence of fluorescence signals.

[0213] This specification demonstrates:

[0214] (a) a process to quantitate viral load in real time using RT-PCR,as described herein;

[0215] (b) a process to quantitate viral load of a Flaviviridae in ahost, including BVDV and HCV, in a host in real time using the RT-PCR,as described herein;

[0216] (c) a process to quantitate viral load of BVDV in a MDBK cellline or a host sample in real time using the RT-PCR, as describedherein;

[0217] (d) a probe molecule designed to fluoresce upon exonucleolyticdegradation and to be complementary to the BVDV NADL NS5B region, asdescribed herein; and

[0218] (e) a probe molecule with a sequence of5′-6-fam-AAATCCTCCTAACAAGCGGGTTCCAGG-tamara-3′ (Sequence ID No 1) andprimers with a sequence of sense: 5′-AGCCTTCAGTTTCTTGCTGATGT-3′(Sequence ID No 2) and antisense: 5′-TGTTGCGAAAGCACCAACAG-3′ (SequenceID No 3);

[0219] (f) a process to quantitate viral load of HCV in a host derivedsample or a cell line in real time using the RT-PCR, as describedherein;

[0220] (g) a probe molecule designed to fluoresce upon exonucleolyticdegradation and to be complementary to the HCV 5′-uncoding region, asdescribed herein; and

[0221] (h) a probe molecule designed to fluoresce upon exonucleolyticdegradation and to be complementary to the HCV coding region, asdescribed herein; and

[0222] (i) a probe molecule designed to fluoresce upon exonucleolyticdegradation and to be complementary to the HCV 3′-uncoding region, asdescribed herein; and

[0223] (j) a probe molecule with a sequence of5′-6-fam-CCTCCAGGACCCCCCCTCCC-tamara-3′ (Sequence ID No 4) and primerswith a sequence of sense: 5′-AGCCATGGCGTTAGTA(T/C)GAGTGT-3′ (Sequence IDNo 5) and antisense: 5′-TTCCGCAGACCACTATGG-3′ (Sequence ID No 6).

BRIEF DESCRIPTION OF THE FIGURES

[0224]FIG. 1 is an illustration of the increase in plaque forming unitswith increasing concentration of bovine viral diarrhea virus (“BVDV”) incell culture as described in Example 51. FIG. 1 establishes that themethod of Example 51 provides reliable quantification of BVDV over afour log PFU/mL of virus.

[0225]FIG. 2 is an illustration of the BVDV replication cycle in MDBKcells to determine the optimal harvesting time (in hours post infectionversus the log of plaque forming units (“PFU”), i.e. 22 hours afterinfection, which roughly corresponds to approximately one replicationcycle, where the amount of virus produced is equal to the amount ofvirus inoculated into the cell, as described in Example 52.

[0226]FIG. 3 is a bar chart graph showing the ability of certain testcompounds to inhibit the number of plaque forming units, as described inExample 40 against BVDV.

[0227]FIG. 4 is a line graph illustrating that the prevention ofcytotoxicity of a “carba-sugar” nucleoside in CEM cells (human T-celllymphoma) and in SUDHL-1 cells (human anaplastic T-cell lymphoma cellline) can be accomplished by co-administration of natural nucleosides,namely cytidine and uridine.

[0228]FIG. 5 provides the structure of various non-limiting examples ofnucleosides of the present invention, as well as the known nucleoside,ribavirin, which is used as a comparative example in the text.

DETAILED DESCRIPTIlON OF THE INVENTION

[0229] The present invention provides a nucleoside of the generalformula (I)-(XXIII) or its pharmaceutically acceptable salt or prodrugfor the treatment of a host infected with a virus belonging to theFlaviviridae, the Orthomyxoviridae, or the Paramyxoviridae family.Alternatively, the nucleoside of the general formula (I) (XXIII) or itspharmaceutically acceptable salt or prodrug can be used for thetreatment of abnormal cellular proliferation.

[0230] In one embodiment, a method for the treatment or prophylaxis ofan antiviral or antiproliferative agent, for example for the treatmentor prophylaxis of a viral infections, including Flaviviridae infections,including hepatitis C infection, influenza virus infection, includinginfluenza A (such as H1N1 and H3N2) and influenza B and RSV, as well asabnormal cellular proliferation that includes the administration of ananti-virally or anti-proliferatively effective amount of a nucleoside ofthe present invention, or its pharmaceutically acceptable salt orprodrug thereof is provided.

[0231] In another embodiment, a method for the treatment or prophylaxisof an antiviral or antiproliferative agent, for example for thetreatment or prophylaxis of a Flaviviridae infection that includes theadministration of an antivirally amount of a nucleoside of the presentinvention, or its pharmaceutically acceptable salt or prodrug thereof inthe manufacture of a medicament for treatment is provided.

[0232] In another embodiment, a method for the treatment or prophylaxisof an antiviral or antiproliferative agent, for example for thetreatment or prophylaxis of an Influenza virus infection that includesthe administration of an antivirally effective amount of a nucleoside ofthe present invention, or its pharmaceutically acceptable salt orprodrug thereof in the manufacture of a medicament for treatment isprovided.

[0233] In another embodiment, a method for the treatment or prophylaxisof an antiviral or antiproliferative agent, for example for thetreatment or prophylaxis of a RSV infection that includes theadministration of an antivirally effective amount of the presentinvention, or its pharmaceutically acceptable salt or prodrug thereof inthe manufacture of a medicament for treatment is provided.

[0234] In another embodiment, a method for the treatment or prophylaxisof an antiviral or antiproliferative agent, for example for thetreatment or prophylaxis of a disease characterized by abnormal cellularproliferation that includes the administration of ananti-proliferatively effective amount of a nucleoside of the presentinvention.

[0235] In another embodiment, the invention is the use of one of thecompounds described herein in the manufacture of a medicament for thetreatment of a viral infection or abnormal cellular proliferation, asprovided herein.

[0236] In another embodiment, the invention is the use of one of thecompounds described herein in the treatment of a host exhibiting a viralinfection or abnormal cellular proliferation, as provided herein.

[0237] In another embodiment, a pharmaceutical composition that includesan antivirally or anti-proliferatively effective amount of a nucleosideof the present invention, or its pharmaceutically acceptable salt orprodrug thereof together with a pharmaceutically acceptable carrier ordiluent according to the present invention is provided.

[0238] In another embodiment, a pharmaceutical composition with anucleoside of the present invention, or its pharmaceutically acceptablesalt or prodrug thereof in combination with one or more otherantivirally or anti-proliferatively effective agents is provided.

[0239] In another embodiment, a process for the preparation of thenucleosides of the present invention, and its pharmaceuticallyacceptable salt and prodrug thereof is provided.

[0240] In an additional embodiment, a method of treating a mammal havinga virus-associated disorder which comprises administering to the mammala pharmaceutically effective amount of a nucleoside of the presentinvention, or their pharmaceutically acceptable salts or prodrugsthereof, is provided.

[0241] In an additional embodiment, a method of treating a mammal havingdisorder associated with abnormal cellular proliferation, whichcomprises administering to the mammal a pharmaceutically effectiveamount of a nucleoside of the present invention, or theirpharmaceutically acceptable salts or prodruigs thereof, is provided.

[0242] In particular, the invention includes the described compounds inmethods for treating or preventing, or uses for the treatment orprophylaxis of, or uses in the manufacture of a medicament forfollowing:

[0243] (a) a Flaviviridae infection, including all members of theHepacivirus genus (HCV), Pestivirus genus (BVDV, CSFV, BDV), orFlavivirus genus (Dengue virus, Japanese encephalitis virus group(including West Nile Virus), and Yellow Fever virus);

[0244] (b) an Orthomyxoviridae infection, including all members of theInfluenza A, B genus, in particular influenza A and all relevantsubtypes—including H1N1 and H3N2-and Influenza B;

[0245] (c) a Paramyxoviridae infection, including Respiratory SyncytialVirus (RSV) infection; and

[0246] (d) abnormal cellular proliferation, including malignant tumors.

[0247] I. Compounds of the Invention

[0248] In one embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a β-D nucleoside of the general formula (I) or(II):

[0249] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0250] each D is hydrogen, alkyl, acyl, monophosphate, diphosphate,triphosphate, monophosphate ester, diphosphate ester, triphosphateester, phospholipid or amino acid, though preferably hydrogen;

[0251] each W¹ and w² is independently CH or N;

[0252] each X¹ and X² is independently hydrogen, halogen (F, Cl, Br orI), NH₂, NHR⁴, NR⁴ R^(4′), NHOR⁴, NR⁴NR^(4′)R^(4″), OH, OR⁴, SH or SR⁴;

[0253] each Y¹ is O, S or Se;

[0254] each Z is CH₂ or NH;

[0255] each R¹ and R^(1′) is independently hydrogen, lower alkyl, loweralkenyl, lower alkynyl, aryl, alkylaryl, halogen (F, Cl, Br or I), NH₂,NHR⁵, NR⁵R⁵, NHOR⁵, NR⁵NHR^(5′), NR⁵NR^(5′)R^(5″), OH, OR⁵, SH, SR⁵,NO₂, NO, CH₂O H, CH₂OR⁵, CO₂H, CO₂R⁵, CONH₂, CONHR⁵, CONR⁵R^(5′) or CN;

[0256] each R² and R^(2′) independently is hydrogen or halogen (F, Cl,Br or I), OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, CH═CH₂, CN, CH₂NH₂, CH₂OH,CO₂H.

[0257] each R³ and R^(3′) independently is hydrogen or halogen (F, Cl,Br or I), OH, SH, OCH₃, SCH₃, NH₂, NHCH₃, CH₃, C₂H₅, CH═CH₂, CN, CH₂NH₂,CH₂O H, CO₂H.

[0258] each R⁴, R^(4′), R^(4″), R⁵, R^(5′) and R^(5″) independently ishydrogen, lower alkyl, lower alkenyl, aryl, or arylalkyl such asunsubstituted or substituted phenyl or benzyl;

[0259] such that for each nucleoside of the general formula (I) or (II),at least one of R² and R^(2′) is hydrogen and at least one of R³ andR^(3′) is hydrogen.

[0260] In another embodiment of the invention, anti-virally oranti-proliferatively effective nucleoside is a β-L nucleoside of thegeneral formula (III) or (IV):

[0261] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0262] each D, W¹, W², X¹, X², Y¹, Z, R¹, R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously;

[0263] such that for each nucleoside of the general formula (III) or(IV), at least one of R² and R^(2′) is hydrogen and at least one of R³and R^(3′) is hydrogen.

[0264] In one embodiment of the invention, the anti-virally oranti-proliferatively effective nucleoside is a β-D-carba-sugarnucleoside of the general formula (V) to (VII):

[0265] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0266] D, W¹, W², X¹, X², Y¹, Z, R¹, R^(1′), R², R^(2′), R³ and R^(3′)is the same as defined previously;

[0267] such that for each nucleoside of the general formula (V) or (VI),at least one of R² and R^(2′) is hydrogen and at least one of R³ andR^(3′) is hydrogen.

[0268] In one embodiment, anti-virally or anti-proliferatively effectivenucleoside is a L-carba-sugar nucleoside of the general formula (VIII)to (X):

[0269] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0270] each D, W¹, W², X¹, X², Y¹, Z, R¹, R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously;

[0271] such that for each nucleoside of the general formula (VIII) or(IX), at least one of R² and R^(2′) is hydrogen and at least one of R³and R^(3′) is hydrogen.

[0272] In further embodiment of the invention, the anti-virally oranti-proliferatively effective β-D or β-L-nucleoside is of the generalformula (XI) or (XII), respectively:

[0273] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0274] each D, W¹, W², X¹, X², Y¹, Z, R¹, R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously;

[0275] each Z¹ and Z² independently is O, S, NR⁶ or Se;

[0276] each R⁶ is hydrogen, lower alkyl or lower acyl.

[0277] In a further embodiment of this invention, the anti-virally oranti-proliferatively effective β-D or β-L-nucleoside, though preferablyβ-D, is of the general formula (XIII):

[0278] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0279] each D, R¹, R^(1′), R², R^(2′), R³ and R^(3′) is the same asdefined previously;

[0280] each Y² is O, S, NH or NR⁷;

[0281] each Y³ is O, S, NH or NR⁸;

[0282] each X³ is OR⁹ or SR⁹; and

[0283] each R⁷, R⁸ and R⁹ is hydrogen, lower alkyl of C₁-C₆, arylalkylor aryl;

[0284] such that for each nucleoside of the general formula (XIII-d), atleast one of R² and R^(2′) is hydrogen and at least one of R³ and R³ ishydrogen.

[0285] In another embodiment, the anti-virally or anti-proliferativelyeffective is a β-D or β-L-nucleoside, though preferably β-D, resultingfrom the addition of a small molecule, such as alkyl hypochlorite, alkylhypobromite, hypobromous acid or acyl halide to an appropriatepyrimidine nucleoside, forming a nucleoside of the formula (XIV):

[0286] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0287] each D, X¹, Y¹, Z¹, R², R^(2′), R³ and R^(3′) is the same asdefined previously;

[0288] each L¹ is hydrogen, Cl or Br;

[0289] each Z³ can be O or CH₂.

[0290] In another embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a dimeric nucleoside (each nucleoside being ineither the β-D or β-L configuration) of general formula (XV), in whichthe two nucleosides are linked through a disulfide bond:

[0291] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0292] each D, W¹, W², X¹, Y¹, Z³, R¹, R^(1′), R², R^(2′), R³ and R^(3′)is the same as defined previously.

[0293] In one embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a β-D or β-L C-nucleoside of the general formula(XVI):

[0294] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0295] each D, W¹, X¹, X², Y¹, Z, R¹, R², R^(2′), R³ and R^(3′) is thesame as defined previously;

[0296] each W³ is independently N, CH or CR¹;

[0297] each W⁴and W⁵ is independently N, CH, CX¹ or CR^(1′); and

[0298] each Z⁴ and Z⁵ is independently NH or C(═Y¹);

[0299] such that if Z⁴ and Z⁵ are covalently bound, then Z⁴ is notC(═Y¹) when Z⁵ is C(═Y¹); and

[0300] there are no more than three ring-nitrogens.

[0301] In one embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a β-D or β-L-branched-chain sugar nucleoside ofthe general formula (XVII):

[0302] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0303] each D, W¹, W², X¹, X¹, Y¹, Z³, R¹, R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously;

[0304] each X⁴ and X⁵ is independently hydrogen, halogen (F, Cl, Br orI), N₃, NH₂, NHR⁸, NR⁸R^(8′), OH, OR SH or SR⁸; and

[0305] each R⁸ and R^(8′) is independently hydrogen, lower alkyl, loweralkenyl, aryl or arylalkyl, such as an unsubstituted or substitutedphenyl or benzyl;

[0306] such that for each nucleoside of the general formula (XVII-a) or(XVII-b), X⁴ is not OH or OR⁸.

[0307] In one embodiment, the anti-virally or anti-proliferativelyeffective nucleoside is a α-D or α-L-nucleoside of the general formula(XVIII):

[0308] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0309] each D, W¹, W², X¹, X², Y¹, R¹, R^(′), R², R^(2′), R³ and R^(3′)is the same as defined previously;

[0310] In a sub-embodiment of the present invention, the anti-virally oranti-proliferatively effective β-D or β-L nucleoside is of the formula(XIX):

[0311] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0312] each D, R¹, R⁴ and R^(4′) is the same as defined previously;

[0313] each R⁹ is hydrogen, halogen (F, Cl, Br or I) or OP³;

[0314] each P¹ is hydrogen, lower alkyl, lower alkenyl, aryl, arylalkyl(such as an unsubstituted or substituted phenyl or benzyl), OH, OR⁴,NH₂, NHR⁴ or NR⁴R^(4′); and

[0315] each P² and P³ is independently hydrogen, alkyl, acyl, -Ms, -Ts,monophosphate, diphosphate, triphosphate, mono-phosphate ester,diphosphate ester, triphosphate ester, phospholipid or amino acid,though preferably hydrogen.

[0316] In a particular sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside ofthe formula (XIX) is the following:

[0317] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0318] each D and P² is the same as defined previously. In a preferredembodiment, D and P² are independently hydrogen.

[0319] In another sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside isof the formula (XX):

[0320] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0321] each D, P¹, P², P³, R¹, R⁴, R^(4′) and R⁹ is the same as definedpreviously.

[0322] In another sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside isof the formula (XXI):

[0323] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0324] each D, P¹, P², P³, R¹, R⁴ and R^(4′) is the same as definedpreviously.

[0325] In a particular sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective E-D or β-L nucleoside ofthe formula (XXI) is the following:

[0326] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0327] each D, P² and P³ is the same as defined previously. In apreferred embodiment, D, P² and P³ are independently hydrogen.

[0328] In another embodiment, N-hydroxycytosine is used as the baseattached to any of the sugar or carba-sugar moieties described in thisapplication, as if each were fully described a separate specificembodiment.

[0329] In another sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside isof the formula (XXII):

[0330] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0331] each D, P¹ and R¹ is the same as defined previously. In apreferred embodiment, D and P² are independently hydrogen.

[0332] In a particular sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside,though preferably β-L, of the formula (XXII) is the following:

[0333] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0334] D is the same as defined previously, and preferably H.

[0335] In another sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside isof the formula (XXIII):

[0336] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0337] each D, P¹, P², P³, R¹, R⁴ and R^(4′) is the same as definedpreviously.

[0338] In a particular sub-embodiment of the present invention, theanti-virally or anti-proliferatively effective β-D or β-L nucleoside ofthe formula (XXIII) is the following:

[0339] or its pharmaceutically acceptable salt or prodrug thereof,wherein:

[0340] each D, P² and P³ is the same as defined previously. In apreferred embodiment, D, P² and P³ are independently hydrogen.

[0341] In a preferred embodiment, the β-D and β-L nucleosides of generalformula (I-a) and (III-a) are represented by the non-limiting examplesprovided in Table 1. TABLE 1

ID X¹ Y¹ R¹ R^(1′) R² R^(2′) R³ R^(3′) AA NH₂ O H H OH H H OH AB NH₂ O HH OH H H I AC NH₂ O H H OH H H Cl AD NH₂ O H H OH H H Br AE NH₂ O H H OHH H S—CN AF NH₂ O H H OH H H N₃ AG NH₂ O H H H Cl H OH AH NH₂ O H H H BrH OH AI NH₂ O H H H OH Br H AJ NH₂ O H H H OH H H AK NH₂ O H H H OH O—MsH AL NH₂ O H H H OH O—Ts H AM NH₂ O H H O—Ms H H OH AN NH₂ O H H Cl H HOH AO NH₂ O D D OH H H OH AP NH₂ O F H OH H H OH AQ NH₂ O F H H OH H OHAR NH₂ O F H H OH H H AS NH₂ O F H H OH Cl H AT NH₂ O F H H OH Br H AUNH₂ O F H H Cl H OH AV NH₂ O F H H OH O—Ts H AW NH₂ O F H H OH O—Ms H AXNH₂ O Cl H H OH O—Ms H AY NH₂ O Br H H OH O—Ms H AZ NH₂ O Br H H OH O—TsH BA NH₂ O Br H H OH Cl H BB NH₂ O Br H H OH H OH BC NH₂ O Br H OH H HOH BD NH₂ O I H H OH O—Ms H BE NH₂ O I H H OH Br H BF NH₂ O I H H OHO—Ts H BG NH₂ O I H H Cl H OH BH NH₂ O I H Br H H OH BI NH₂ O OH H OH HH OH BJ NH₂ O NH₂ H H OH H OH BK NH₂ O CH₃ H H OH Cl H BL NH₂ NH H H OHH H OH BM NH₂ S H H H Se-phenyl H H BN NH-(2-Ph—Et) O H H OH H H OH BONH—COCH₃ O H H OH H H OH BP NH—NH₂ O H H OH H H OH BQ NH—NH₂ O F H OH HH OH BR NH—NH₂ O CH₃ H H OH H OH BS NH—OH O H H H OH H OH BT NH—OH O F HH OH H OH BU NH—OH O Br H H OH H OH BV NH—OH O I H H OH H OH BW NH—OH OH H OH H H OH BX OH O OH H OH H H OH BY OH O NH₂ H H OH H OH BZ OH O F HOH H H OH CA OH O F H H O—Ts H OH CB OH O F H H O—Ms H O—Ms CC OH O F HH OH H OH CD OH O F H H OH H O—Ts CE OH O F H H H H OH CF O—Et O H H HO—Bz H O—Bz CG S—CH₃ O H H H F H OH CH SH O H H H OH H OH CI SH O F H HOH H OH CJ N₃ O H H H H H H CK NH-(2-Ph—Et) O H H H OH H OH CL OH O OH HH OH H OH CM OH O H H H OH H H

[0342] In a preferred embodiment, the β-D and β-L nucleosides of generalformula (I-b) and (III-b) are represented by the non-limiting examplesprovided in Table 2. TABLE 2

ID X¹ X² W¹ R² R^(2′) R³ R^(3′) DA OH NH₂ N H OH H OH DB OH NH₂ CH F H HOH DC NH-cyclohexyl H CH H H H H DD NH₂ H CH H OH H F DE NH₂ H CH H H HH DF NH₂ NH₂ N H OH H OH DG NH₂ NH₂ CH H OH H OH DH Cl H CH F H H H DICl I CH H O—Ac H O—Ac DJ Cl H CH H OH H OH DK NH₂ H CH H OH H H DL Cl HCH H OH H H

[0343] In a preferred embodiment, the β-D and β-L nucleosides of generalformula (II-a) and (IV-a) are represented by the non-limiting examplesprovided in Table 3. TABLE 3

ID X¹ Y¹ R¹ R^(1′) R² R³ EA NH—Bz-(m-NO₂) O F H H H EB NH—Bz-(o-NO₂) O FH H H EC NH₂ O F H F H

[0344] In a preferred embodiment, the β-D and β-L nucleosides of generalformula (II-b) and (IV-b) are represented by the non-limiting examplesprovided in Table 4. TABLE 4

ID X¹ X² W¹ R² R³ FA Cl H CH F H FB OH H CH H H FC NH₂ F CH H H FD NH₂ FCH F H FE NH₂ H CH H H FF OH NH₂ CH H H FG OH H CH H H

[0345] In a preferred embodiment, the β-D and β-L nucleosides of generalformula (V-a) and (VIII-a) are represented by the non-limiting examplesprovided in Table 5. TABLE 5

ID X¹ Y¹ R¹ R^(1′) R² R^(2′) R³ R^(3′) GA NH₂ O F H H OH H OH GB OH HCH₃ H H H H H GC OH O H H H H H H GD NH₂ O H H H OH H OH GE NH₂ O H H HH H H GF OH O F H H OH H OH GG NH₂ O I H H H H H GH NH₂ O I H H OH H OHGI NH₂ O Cl H H OH H OH

[0346] In a preferred embodiment, the β-D and β-L nucleosides of generalformula (VII-a) and (X-a) are represented by the non-limiting examplesprovided in Table 6. TABLE 6

ID X¹ Y¹ R¹ R^(1′) R² R^(2′) R³ R^(3′) HA NH₂ O H H H OH H OH HB NH₂ O FH H OH H OH HC NH—OH O H H H OH H OH

[0347] In a preferred embodiment, the β-D and β-L nucleosides of generalformula (VII-b) and (X-b) are represented by the non-limiting examplesprovided in Table 7. TABLE 7

ID X¹ X² W¹ R² R^(2′) R³ R^(3′) IA NH₂ H CH H OH H OH

[0348] In a preferred embodiment, the β-D or β-L nucleosides of generalformula (XI-a) or (XIII-a) are represented by the non-limiting examplesprovided in Table 8. TABLE 8

ID X¹ Y¹ Z¹ Z² R¹ R^(1′) JA NH₂ O O O H H JB NH₂ O O S F H JC NH₂ O O OF H

[0349] In a preferred embodiment, the β-L nucleosides of general formula(XII-b) are represented by the non-limiting examples provided in Table9. TABLE 9

ID X¹ X² W¹ Z¹ Z² KA Cl H CH O S KB Cl NH₂ CH O S KC NH₂ F CH O S KD OHH CH O O

[0350] In a preferred embodiment, the β-D nucleosides of general formula(XIII-a) are represented by the non-limiting examples provided in Table10. TABLE 10

ID Y² Y³ R¹ R^(1′) R² R^(2′) R³ R^(3′) LA O O F H H OH H OH

[0351] In a preferred embodiment, the β-D nucleosides of general formula(XIII-c) are represented by the non-limiting examples provided in Table11. TABLE 11 [XIII-c]

ID Y² Y³ R¹ R^(1′) R³ R^(3′) MA O O F H H OH MB O O F H H O—Ms MC NH O HH H O—Ms MD NH O H H H O—Ac ME NH O H H H OH MF NH O F H H OH MG NH O FH H O—Ac

[0352] In a preferred embodiment, the β-D nucleosides of general formula(XIII-d) are represented by the non-limiting examples provided in Table12. TABLE 12 [XIII-d]

ID Y² X³ R¹ R^(1′) R² R^(2′) R³ R^(3′) NA O O—CH₃ H H H O—Ac H O—Ac

[0353] In a preferred embodiment, the β-D nucleosides of general formula(XIV) are represented by the non-limiting examples provided in Table 13.TABLE 13 [XIV]

ID X¹ Y¹ R¹ R^(1′) R² R^(2′) R³ R^(3′) L¹ L² OA NH₂ O NH—OH OH OH H H OHH OH OB OH O O F H OH H OH Cl O—CH₃ OC OH O O H H OH H OH Br O—CH₃ OD OHO O F H OH H OH Br O—COCH₃ OE OH O O F H OH H OH Br O—CH₃ OF OH O O F HOH H OH Br O—Et OG OH O O Cl H OH H OH Br O—CH₃

[0354] In a preferred embodiment, the nucleosides of general formula(XV-a) are represented by the non-limiting examples provided in Table14. TABLE 14 [XV-a]

ID Y¹ Z³ R¹ R^(1′) R² R^(2′) R³ R^(3′) PA O O H H H OH H OH

[0355] In a preferred embodiment, the nucleosides of general formula(XV-b) are represented by the non-limiting examples provided in Table15. TABLE 15 [XV-b]

ID X¹ W¹ Z³ R² R^(2′) R³ R^(3′) QA NH₂ CH O H OH H OH

[0356] In a preferred embodiment, the nucleosides of general formula(XVI-a) are represented by the non-limiting examples provided in Table16. TABLE 16 [XVI-a]

ID W³ Z⁴ W⁵ W⁴ Z⁵ R² R^(2′) R³ R^(3′) RA CH NCH₃ C—OH N C═O H OH H O—TsRB CH NH C—NH₂ N C═O H OH H OH RC CH NH C—NHAc N C═O H OH H OH RD CH NHC—OH N C═O H OH H OH RE CH NCH₃ C—NH₂ N C═O H OH H OH RF CH NH C—NHBz NC═O H OH H OH RG CH C═O C—NH₂ C—SH NH H OH H OH RH CH NH C—OH N C═O H ClH OH RI CH NH C—NH₂ N C═O H Br H OH

[0357] In a preferred embodiment, the nucleosides of general formula(XVI-c) are represented by the non-limiting examples provided in Table17. TABLE 17 [XVI-c]

ID W³ Z⁴ Z⁵ W⁴ R² R^(2′) R³ R^(3′) SA CH N—CH₃ C═O N H OH H O—Ac

[0358] In a preferred embodiment, the nucleosides of general formula(XVI-d) are represented by the non-limiting examples provided in Table18. TABLE 18 [XVI-d]

ID W³ Z⁴ Z⁵ W⁴ R³ R^(3′) TA CH N C═NH N H OH

[0359] In a preferred embodiment, the nucleosides of general formula(XVI-f) are represented by the non-limiting examples provided in Table19. TABLE 19 [XVI-f]

ID X¹ X² W¹ R² R^(2′) R³ R^(3′) UA NH₂ H N H OH H OH

[0360] In a preferred embodiment, the nucleosides of general formula(XVII-d) are represented by the non-limiting examples provided in Table20. TABLE 20 [XVII-d]

ID X¹ X² W¹ X⁴ X⁵ VA NH₂ F CH H OH

[0361] In one embodiment, the nucleoside has an EC₅₀ (effectiveconcentration to achieve 50% viral inhibition) when tested in anappropriate cell-based assay, of less than 15 micromolar, and moreparticularly, less than 10 or 5 micromolar. In a preferred embodiment,the nucleoside is enantiomerically enriched.

[0362] II. Stereoisomerism and Polymorphism

[0363] Compounds of the present invention having a chiral center mayexist in and be isolated in optically active and racemic forms. Somecompounds may exhibit polymorphism. The present invention encompassesracemic, optically-active, polymorphic, or stereoisomeric form, ormixtures thereof, of a compound of the invention, which possess theuseful properties described herein. The optically active forms can beprepared by, for example, resolution of the racemic form byrecrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase or by enzymatic resolution.

[0364] As shown below, a nucleoside contains at least two criticalchiral carbon atoms (*). In general, the substituents on the chiralcarbons [the specified purine or pyrimidine base (referred to as the C1substituent when using the sugar ring intermediate numbering) and CH₂O H(referred to as the C4 substituent)] of the nucleoside can be either cis(on the same side) or trans (on opposite sides) with respect to thesugar ring system. Both the cis and trans racemates consist of a pair ofoptical isomers. Hence, each compound has four individual stereoisomers.The four stereoisomers are represented by the following configurations(when orienting the sugar moiety in a horizontal plane such that the−O—moiety is in back): (1) cis, with both groups “up”, which is referredto as β-D; (2) the mirror image, i.e., cis, with both groups “down”,which is the mirror image is referred to as β-L; (3) trans with the C4substituent “up” and the C1 substituent “down” (referred to as α-D); and(4) trans with the C4 substituent “down” and the C1 substituent “up”(referred to as α-L). The two cis enantiomers together are referred toas a racemic mixture of β-enantiomers, and the two trans enantiomers arereferred to as a racemic mixture of α-enantiomers.

[0365] The four possible stereoisomers of the claimed compounds areillustrated below.

[0366] III. Definitions

[0367] The term “alkyl,” as used herein, unless otherwise specified,refers to a saturated straight, branched, or cyclic, primary, secondary,or tertiary hydrocarbon, including but not limited to those of C₁ toC₁₆, and specifically includes methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl,neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The alkylgroup can be optionally substituted with one or more moieties selectedfrom the group consisting of alkyl, halo, haloalkyl, hydroxyl, carboxyl,acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino,dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, azido, thiol,imine, sulfonic acid, sulfate, sulfonyl, sulfanyl, sulfinyl, sulfamonyl,ester, carboxylic acid, amide, phosphonyl, phosphinyl, phosphoryl,phosphine, thioester, thioether, acid halide, anhydride, oxime,hydrozine, carbamate, phosphonic acid, phosphate, phosphonate, or anyother viable functional group that does not inhibit the pharmacologicalactivity of this compound, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al., Protective Groups in Organic Synthesis, John Wileyand Sons, Second Edition, 1991, hereby incorporated by reference.

[0368] The term “lower alkyl,” as used herein, and unless otherwisespecified, refers to a C₁ to C₄ saturated straight, branched, or ifappropriate, a cyclic (for example, cyclopropyl) alkyl group, includingboth substituted and unsubstituted forms.

[0369] The term “alkylene” or “alkenyl” refers to a saturatedhydrocarbyldiyl radical of straight or branched configuration, includingbut not limited to those that have from one to ten carbon atoms.Included within the scope of this term are methylene, 1,2-ethane-diyl,1,1-ethane-diyl, 1,3-propane-diyl, 1,2-propane-diyl, 1,3-butane-diyl,1,4-butane-diyl and the like. The alkylene group or other divalentmoiety disclosed herein can be optionally substituted with one or moremoieties selected from the group consisting of alkyl, halo, haloalkyl,hydroxyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives,alkylamino, azido, dialkylamino, arylamino, alkoxy, aryloxy, nitro,cyano, sulfonic acid, thiol, imine, sulfonyl, sulfanyl, sulfinyl,sulfamonyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl,phosphoryl, phosphine, thioester, thioether, acid halide, anhydride,oxime, hydrozine, carbamate, phosphonic acid, phosphonate, or any otherviable functional group that does not inhibit the pharmacologicalactivity of this compound, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al., Protective Groups in Organic Synthesis, John Wileyand Sons, Second Edition, 1991, hereby incorporated by reference.

[0370] The term “aryl,” as used herein, and unless otherwise specified,refers to phenyl, biphenyl, or naphthyl, and preferably phenyl. The termincludes both substituted and unsubstituted moieties. The aryl group canbe substituted with one or more moieties selected from the groupconsisting of bromo, chloro, fluoro, iodo, hydroxyl, azido, amino,alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991.

[0371] The term “aralkyl,” as used herein, and unless otherwisespecified, refers to an aryl group as defined above linked to themolecule through an alkyl group as defined above. The term “alkaryl” or“alkylaryl” as used herein, and unless otherwise specified, refers to analkyl group as defined above linked to the molecule through an arylgroup as defined above. In each of these groups, the alkyl group can beoptionally substituted as describe above and the aryl group can beoptionally substituted with one or more moieties selected from the groupconsisting of alkyl, halo, haloalkyl, hydroxyl, carboxyl, acyl, acyloxy,amino, amido, azido, carboxyl derivatives, alkylamino, dialkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, thiol, imine,sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester, carboxylic acid, amide,phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether,acid halide, anhydride, oxime, hydrozine, carbamate, phosphonic acid,phosphonate, or any other viable functional group that does not inhibitthe pharmacological activity of this compound, either unprotected, orprotected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference. Specifically included within the scope of theterm aryl are phenyl; naphthyl; phenylmethyl; phenylethyl;3,4,5-trihydroxyphenyl; 3,4,5-trimethoxyphenyl; 3,4,5-triethoxy-phenyl;4-chlorophenyl; 4-methylphenyl; 3,5-di-tertiarybutyl-4-hydroxyphenyl;4-fluorophenyl; 4-chloro-1-naphthyl; 2-methyl-1-naphthylmethyl;2-naphthylmethyl; 4-chlorophenylmethyl; 4-t-butylphenyl;4-t-butylphenylmethyl and the like.

[0372] The term “alkylamino” or “arylamino” refers to an amino groupthat has one or two alkyl or aryl substituents, respectively.

[0373] The term “halogen,” as used herein, includes fluorine, chlorine,bromine and iodine.

[0374] The term “enantiomerically enriched” is used throughout thespecification to describe a nucleoside which includes at least about95%, preferably at least 96%, more preferably at least 97%, even morepreferably, at least 98%, and even more preferably at least about 99% ormore of a single enantiomer of that nucleoside. When a nucleoside of aparticular configuration (D or L) is referred to in this specification,it is presumed that the nucleoside is an enantiomerically enrichednucleoside, unless otherwise stated.

[0375] The term “host,” as used herein, refers to a unicellular ormulticellular organism in which the virus can replicate, including celllines and animals, and preferably a human. Alternatively, the host canbe carrying a part of the viral genome, whose replication or functioncan be altered by the compounds of the present invention. The term hostspecifically refers to infected cells, cells transfected with all orpart of the viral genome and animals, in particular, primates (includingchimpanzees) and humans. Relative to abnormal cellular proliferation,the term “host” refers to unicellular or multicellular organism in whichabnormal cellular proliferation can be mimicked. The term hostspecifically refers to cells that abnormally proliferate, either fromnatural or unnatural causes (for example, from genetic mutation orgenetic engineering, respectively), and animals, in particular, primates(including chimpanzees) and humans. In most animal applications of thepresent invention, the host is a human patient. Veterinary applications,in certain indications, however, are clearly anticipated by the presentinvention (such as bovine viral diarrhea virus in cattle, hog choleravirus in pigs, and border disease virus in sheep).

[0376] The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a compound which, upon administration to a patient, providesthe active compound. Pharmaceutically acceptable salts include thosederived from pharmaceutically acceptable inorganic or organic bases andacids. Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart. Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on afunctional moiety of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated, dephosphorylated to produce theactive compound.

[0377] IV. Pharmaceutically Acceptable Salts and Prodrugs

[0378] In cases where compounds are sufficiently basic or acidic to formstable nontoxic acid or base salts, administration of the compound as apharmaceutically acceptable salt may be appropriate. Pharmaceuticallyacceptable salts include those derived from pharmaceutically acceptableinorganic or organic bases and acids. Suitable salts include thosederived from alkali metals such as potassium and sodium, alkaline earthmetals such as calcium and magnesium, among numerous other acids wellknown in the pharmaceutical art. In particular, examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids, which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, a-ketoglutarate, and a-glycerophosphate.Suitable inorganic salts may also be formed, including, sulfate,nitrate, bicarbonate, and carbonate salts.

[0379] Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

[0380] Any of the nucleosides described herein can be administered as anucleotide prodrug to increase the activity, bioavailability, stabilityor otherwise alter the properties of the nucleoside. A number ofnucleotide prodrug ligands are known. In general, alkylation, acylationor other lipophilic modification of the mono, di or triphosphate of thenucleoside will increase the stability of the nucleotide. Examples ofsubstituent groups that can replace one or more hydrogens on thephosphate moiety are alkyl, aryl, steroids, carbohydrates, includingsugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jonesand N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of thesecan be used in combination with the disclosed nucleosides to achieve adesired effect.

[0381] The active nucleoside can also be provided as a 5′-phosphoetherlipid or a 5′-ether lipid, as disclosed in the following references,which are incorporated by reference herein: Kucera, L. S., N. Iyer, E.Leake, A. Raben, Modest E. K., D. L. W., and C. Piantadosi. 1990. “Novelmembrane-interactive ether lipid analogs that inhibit infectious HIV-1production and induce defective virus formation.” AIDS Res. Hum. RetroViruses. 6:491-501; Piantadosi, C., J. Marasco C. J., S. L.Morris-Natschke, K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq, L. S.Kucera, N. Iyer, C. A. Wallen, S. Piantadosi, and E. J. Modest. 1991.“Synthesis and evaluation of novel ether lipid nucleoside conjugates foranti-HIV activity.” J. Med. Chem. 34:1408.1414; Hosteller, K. Y., D. D.Richman, D. A. Carson, L. M. Stuhmiller, G. M. T. van Wijk, and H. vanden Bosch. 1992. “Greatly enhanced inhibition of human immunodeficiencyvirus type I replication in CEM and HT4-6C cells by 3′-deoxythymidinediphosphate dimyristoylglycerol, a lipid prodrug of 3β-deoxythymidine.”Antimicrob. Agents Chemother. 36:2025.2029; Hosetler, K. Y., L. M.Stuhmiller, H. B. Lenting, H. van den Bosch, and D. D. Richman, 1990.“Synthesis and antiretroviral activity of phospholipid analogs ofazidothymidine and other antiviral nucleosides.” J. Biol. Chem.265:61127.

[0382] Nonlimiting examples of U.S. patents that disclose suitablelipophilic substituents that can be covalently incorporated into thenucleoside, preferably at the 5′-OH position of the nucleoside orlipophilic preparations, include U.S. Pat. Nos. 5,149,794 (Sep. 22,1992, Yatvin et al.); 5,194,654 (Mar. 16, 1993, Hostetler et al.,5,223,263 (Jun. 29, 1993, Hostetler et al.); 5,256,641 (Oct. 26, 1993,Yatvin et al.); 5,411,947 (May 2, 1995, Hostetler et al.); 5,463,092(Oct. 31, 1995, Hostetler et al.); 5,543,389 (Aug. 6, 1996, Yatvin etal.); 5,543,390 (Aug. 6, 1996, Yatvin et al.); 5,543,391 (Aug. 6, 1996,Yatvin et al.); and 5,554,728 (Sep. 10, 1996; Basava et al.), all ofwhich are incorporated herein by reference. Foreign patent applicationsthat disclose lipophilic substituents that can be attached to thenucleosides of the present invention, or lipophilic preparations,include WO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910,WO 94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.

[0383] V. Pharmaceutical Compositions

[0384] Pharmaceutical compositions based upon a β-D or β-L compound offormula (I)-(XXIII) or its pharmaceutically acceptable salt or prodrugcan be prepared in a therapeutically effective amount for treating aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation, optionally in combination with apharmaceutically acceptable additive, carrier or excipient. Thetherapeutically effective amount may vary with the infection orcondition to be treated, its severity, the treatment regimen to beemployed, the pharmacokinetics of the agent used, as well as the patienttreated.

[0385] In one aspect according to the present invention, the compoundaccording to the present invention is formulated preferably in admixturewith a pharmaceutically acceptable carrier. In general, it is preferableto administer the pharmaceutical composition in orally administrableform, but formulations may be administered via parenteral, intravenous,intramuscular, transdermal, buccal, subcutaneous, suppository or otherroute. Intravenous and intramuscular formulations are preferablyadministered in sterile saline. One of ordinary skill in the art maymodify the formulation within the teachings of the specification toprovide numerous formulations for a particular route of administrationwithout rendering the compositions of the present invention unstable orcompromising its therapeutic activity. In particular, a modification ofa desired compound to render it more soluble in water or other vehicle,for example, may be easily accomplished by routine modification (saltformulation, esterification, etc.).

[0386] In certain pharmaceutical dosage forms, the prodrug form of thecompound, especially including acylated (acetylated or other) and etherderivatives, phosphate esters and various salt forms of the presentcompounds, is preferred. One of ordinary skill in the art will recognizehow to readily modify the present compound to a prodrug form tofacilitate delivery of active compound to a targeted site within thehost organism or patient. The artisan also will take advantage offavorable pharmacokinetic parameters of the prodrug form, whereapplicable, in delivering the desired compound to a targeted site withinthe host organism or patient to maximize the intended effect of thecompound in the treatment of Flaviviridae (including HCV),Orthomyxoviridae (including Influenza A and B), Paramyxoviridae(including RSV) infections or conditions related to abnormal cellularproliferation.

[0387] The amount of compound included within therapeutically activeformulations, according to the present invention, is an effective amountfor treating the infection or condition, in preferred embodiments, aFlaviviridae (including HCV), Orthomyxoviridae (including Influenza Aand B), Paramyxoviridae (including RSV) infection or a condition relatedto abnormal cellular proliferation. In general, a therapeuticallyeffective amount of the present compound in pharmaceutical dosage formusually ranges from about 0.1 mg/kg to about 100 mg/kg or more,depending upon the compound used, the condition or infection treated andthe route of administration. For purposes of the present invention, aprophylactically or preventively effective amount of the compositions,according to the present invention, falls within the same concentrationrange as set forth above for therapeutically effective amount and isusually the same as a therapeutically effective amount.

[0388] Administration of the active compound may range from continuous(intravenous drip) to several oral administrations per day (for example,Q.I.D., B.I.D., etc.) and may include oral, topical, parenteral,intramuscular, intravenous, subcutaneous, transdermal (which may includea penetration enhancement agent), buccal and suppository administration,among other routes of administration. Enteric-coated oral tablets mayalso be used to enhance bioavailability and stability of the compoundsfrom an oral route of administration. The most effective dosage formwill depend upon the pharmacokinetics of the particular agent chosen, aswell as the severity, of disease in the patient. Oral dosage forms areparticularly preferred, because of ease of administration andprospective favorable patient compliance.

[0389] To prepare the pharmaceutical compositions according to thepresent invention, a therapeutically effective amount of one or more ofthe compounds according to the present invention is preferably mixedwith a pharmaceutically acceptable carrier according to conventionalpharmaceutical compounding techniques to produce a dose. A carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, e.g., oral or parenteral. In preparingpharmaceutical compositions in oral dosage form, any of the usualpharmaceutical media may be used. Thus, for liquid oral preparationssuch as suspensions, elixirs and solutions, suitable carriers andadditives including water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents and the like may be used. For solid oralpreparations such as powders, tablets, capsules, and for solidpreparations such as suppositories, suitable carriers and additivesincluding starches, sugar carriers, such as dextrose, mannitol, lactoseand related carriers, diluents, granulating agents, lubricants, binders,disintegrating agents and the like may be used. If desired, the tabletsor capsules may be enteric-coated for sustained release by standardtechniques. The use of these dosage forms may significantly impact thebioavailability of the compounds in the patient.

[0390] For parenteral formulations, the carrier will usually comprisesterile water or aqueous sodium chloride solution, though otheringredients, including those that aid dispersion, also may be included.Where sterile water is to be used and maintained as sterile, thecompositions and carriers must also be sterilized. Injectablesuspensions may also be prepared, in which case appropriate liquidcarriers, suspending agents and the like may be employed.

[0391] Liposomal suspensions (including liposomes targeted to viralantigens) may also be prepared by conventional methods to producepharmaceutically acceptable carriers. This may be appropriate for thedelivery of free nucleosides, acyl nucleosides or phosphate esterprodrug forms of the nucleoside compounds according to the presentinvention.

[0392] In particularly preferred embodiments according to the presentinvention, the compounds and compositions are used to treat, prevent ordelay the onset of Flaviviridae (including HCV), Orthomyxoviridae(including Influenza A and B), Paramyxoviridae (including RSV)infections or conditions related to abnormal cellular proliferation.Preferably, to treat, prevent or delay the onset of the infection orcondition, the compositions will be administered in oral dosage form inamounts ranging from about 250 micrograms up to about 1 gram or more atleast once a day, preferably, or up to four times a day. The presentcompounds are preferably administered orally, but may be administeredparenterally, topically or in suppository form.

[0393] The compounds according to the present invention, because oftheir low toxicity to host cells in certain instances, may beadvantageously employed prophylactically to prevent Flaviviridae(including HCV), Orthomyxoviridae (including Influenza A and B),Paramyxoviridae (including RSV) infections or conditions related toabnormal cellular proliferation or to prevent the occurrence of clinicalsymptoms associated with the viral infection or condition. Thus, thepresent invention also encompasses methods for the prophylactictreatment of viral infection, and in particular Flaviviridae (includingHCV), Orthomyxoviridae (including Influenza A and B), Paramyxoviridae(including RSV) infections or of a condition related to abnormalcellular proliferation. In this aspect, according to the presentinvention, the present compositions are used to prevent or delay theonset of a Flaviviridae (including HCV), Orthomyxoviridae (includingInfluenza A and B), Parainyxoviridae (including RSV) infection or acondition related to abnormal cellular proliferation. This prophylacticmethod comprises administration to a patient in need of such treatment,or who is at risk for the development of the virus or condition, anamount of a compound according to the present invention effective foralleviating, preventing or delaying the onset of the viral infection orcondition. In the prophylactic treatment according to the presentinvention, it is preferred that the antiviral or antiproliferativecompound utilized should be low in toxicity and preferably non-toxic tothe patient. It is particularly preferred in this aspect of the presentinvention that the compound that is used should be maximally effectiveagainst the virus or condition and should exhibit a minimum of toxicityto the patient. In the case of Flaviviridae (including HCV),Orthomyxoviridae (including Influenza A and B), Paramyxoviridae(including RSV) infections or conditions related to abnormal cellularproliferation, compounds according to the present invention, which maybe used to treat these disease states, may be administered within thesame dosage range for therapeutic treatment (i.e., about 250 microgramsup to 1 gram or more from one to four times per day for an oral dosageform) as a prophylactic agent to prevent the proliferation of aFlaviviridae (including HCV), Orthomyxoviridae (including Influenza Aand B), Paramyxoviridae (including RSV) infections or conditions relatedto abnormal cellular proliferation, or alternatively, to prolong theonset of a Flaviviridae (including HCV), Orthomyxoviridae (includingInfluenza A and B), Paramyxoviridae (including RSV) infections orconditions related to abnormal cellular proliferation, which manifestsitself in clinical symptoms.

[0394] In addition, compounds according to the present invention can beadministered in combination or alternation with one or more antiviral,anti-HBV, anti-HCV or anti-herpetic agent or interferon, anti-cancer orantibacterial agents, including other compounds of the presentinvention. Certain compounds according to the present invention may beeffective for enhancing the biological activity of certain agentsaccording to the present invention by reducing the metabolism,catabolism or inactivation of other compounds and as such, areco-administered for this intended effect.

[0395] This invention is further illustrated in the following sections.The Experimental Details section and Examples contained therein are setforth to aid in an understanding of the invention. This section is notintended to, and should not be interpreted to, limit in any way theinvention set forth in the claims that follow thereafter.

[0396] VI. Therapies for the Treatment of Flaviviridae Infection

[0397] It has been recognized that drug-resistant variants of virusescan emerge after prolonged treatment with an antiviral agent. Drugresistance most typically occurs by mutation of a gene that encodes foran enzyme used in the viral replication cycle, and most typically in thecase of HCV, the RNA-dependent-RNA polymerase. It has been demonstratedthat the efficacy of a drug against viral infection can be prolonged,augmented, or restored by administering the compound in combination oralternation with a second, and perhaps third, antiviral compound thatinduces a different mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution or other parameterof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous stresses on the virus.

[0398] Examples of agents that have been identified as active againstthe hepatitis C virus, and thus can be used in combination oralternation with one or more nucleosides of general formula (I)-(XXIII)include:

[0399] (a) interferon and ribavirin (Battaglia, A. M. et al. Ann.Pharmacother. 2000, 34, 487; Berenguer, M. et al. Antivir. Ther. 1998, 3(Suppl. 3), 125);

[0400] (b) Substrate-based NS3 protease inhibitors (Attwood et al. PCTWO 98/22496, 1998; Attwood et al. Antiviral Chemistry and Chemotherapy1999, 10, 259,; Attwood et al. German Patent Publication DE 19914474;Tung et al. PCT WO 98/17679), including alphaketoamides andhydrazinoureas, and inhibitors that terminate in an electrophile such asa boronic acid or phosphonate (Llinas-Brunet et. al. PCT WO 99/07734);

[0401] (c) Non-substrate-based inhibitors such as2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,Biochemical and Biophysical Research Communications, 1997, 238, 643 andSudo K. et al. Antiviral Chemistry and Chemotherapy 1998, 9, 186),including RD3-4082 and RD3-4078, the former substituted on the amidewith a 14 carbon chain and the latter processing apara-phenoxyphenylgroup;

[0402] (d) Thiazolidine derivatives which show relevant inhibition in areverse-phase HPLC assay with an NS3/4A fusion protein anti NS5A/5Bsubstrate (Sudo K. et al. Antiviral Research 1996, 32, 9), especiallycompound RD-1-6250, possessing a fused cinnamoyl moiety substituted witha long alkyl chain, RD4 6205 and RD4 6193;

[0403] (e) Thiazolidines and benzanilides identified in Kakiuchi N. etal. J. EBS Letters 421, 217 and Takeshita N. et al. AnalyticalBiochemistry 1997, 247, 242;

[0404] (f) A phenanthrenequinone possessing activity against HCVprotease in a SDS-PAGE and autoradiography assay isolated from thefermentation culture broth of Streptomyces sp., Sch 68631 (Chu M. et al.Tetrahedron Letters 1996, 37, 7229), and Sch 351633, isolated from thefungus Penicillium griscofuluum, which demonstrates activity in ascintillation proximity assay (Chu M. et al., Bioorganic and MedicinalChemistry Letters 9, 1949);

[0405] (g) Selective NS3 inhibitors based on the macromolecule elgin c,isolated from leech (Qasim M. A. et al. Biochemistry 1997, 36,1598);

[0406] (h) HCV helicase inhibitors (Diana G. D. et al, U.S. Pat. No.5,633,358 and Diana G.

[0407] D. et al. PCT WO 97/36554);

[0408] (i) HCV polymerase inhibitors such as nucleotide analogues,gliotoxin (Ferrari R. et al. Journal of Virology 1999, 73, 1649), andthe natural product cerulenin (Lohmann V. et al. Virology 1998, 249,108);

[0409] (j) Antisense phosphorothioate oligodeoxynucleotides (S—ODN)complementary to at least a portion of a sequence of the HCV (Andersonet al. U.S. Pat. No. 6,174,868), and in particular the sequencestretches in the 5′ non-coding region (NCR) (Alt M. et al. Hepatology1995, 22, 707), or nucleotides 326-348 comprising the 3′ end of the NCRand nucleotides 371-388 located in the core coding region of the HCV RNA(Alt M. et al. Archives of Virology 1997, 142, 589 and Galderisi U. etal., Journal of Cellular Physiology 1999, 81:2151);

[0410] (k) Inhibitors of IRES-dependent translation (Ikeda N et al.Japanese Patent Pub. JP-08268890; Kai Y. et al. Japanese PatentPublication JP-10101591);

[0411] (l) Nuclease-resistant ribozymes (Maccjak D. J. et al.,Hepatology 1999, 30, abstract 995);

[0412] (m) Amantadine, such as rimantadine (Smith, Abstract from AnnualMeeting of the American Gastoenterological Association and AASLD, 1996);

[0413] (n) Quinolones, such as ofloxacin, ciprofloxacin and levofloxacin(AASLD Abstracts, Hepatology, October 1994, Program Issue, 20 (4), pt.2,abstract no. 293);

[0414] (o) Nucleoside analogs (Ismaili et al. WO 01/60315; Storer WO01/32153), including 2′-deoxy-L-nucleosides (Watanabe et al. WO01/34618), and 1-(β-L-ribofuranosyl)-1,2,4-triazole-3-carboxamide(levovirin™) (Tam WO 01/46212); and

[0415] (p) Other miscellaneous compounds includingI-amino-alkylcyclohexanes (Gold et al. U.S. Pat. No. 6,034,134), alkyllipids (Chojkier et al. U.S. Pat. No. 5,922,757), vitamin E and otherantioxidants (Chojkier et al. U.S. Pat. No. 5,922,757), squalene, bileacids (Ozeki et al. U.S. Pat. No. 5,846,964),N-(phosphonoacetyl)-L-aspartic acid, (Diana et al. U.S. Pat. No.5,830,905), benzenedicarboxamides (Diana et al. U.S. Pat. No.5,633,388), polyadenylic acid derivatives (Wang et al. U.S. Pat. No.5,496,546), 2′,3′-dideoxyinosine (Yarchoan et al U.S. Pat. No.5,026,687), benzimidazoles (Colacino et al. U.S. Pat. No. 5,891,874),glucamines (Mueller et al. WO 01/08672),substituted-1,5-imino-D-glucitol compounds (Mueller et al. WO 00/47198).

[0416] VII. Therapies for the Treatment of Orthomyxoviridae Infection

[0417] It has been recognized that drug-resistant variants of influenzacan emerge after prolonged treatment with an antiviral agent. Drugresistance most typically occurs by mutation of a gene that encodes foran enzyme used in the viral replication cycle, resulting in antigenicshifts or drifts. It has been demonstrated that the efficacy of a drugagainst influenza infection can be prolonged, augmented, or restored byadministering the compound in combination or alternation with a second,and perhaps third, antiviral compound that induces a different mutationfrom that caused by the principle drug. Alternatively, thepharmacokinetics, biodistribution or other parameter of the drug can bealtered by such combination or alternation therapy. In general,combination therapy is typically preferred over alternation therapybecause it induces multiple simultaneous stresses on the virus.

[0418] Examples of agents that have been identified as active againstthe influenza virus, and thus can be used in combination or alternationwith one or more nucleosides of general formula(I)-(XXIII) include:

[0419] (a) actinomycin D (Barry, R. D. et al. “Participation ofdeoxyribonucleic acid in the multiplication of influenza virus” Nature,1962, 194, 1139-1140);

[0420] (b) amantadine (Van Voris, L. P. et al. “Antivirals for thechemoprophylaxis and treatment of influenza” Semin Respir Infect, 1992,7, 61-70);

[0421] (c) 4-amino- or4-guanidino-2-deoxy-2,3-didehydro-D-N-acetylneuroaminic acid-4-amino- or4-guanidino-Neu 5 Ac2en (von Itzstein, M. et al. “Rational design ofpotent sialidase-based inhibitors of influenza virus replication”Nature, 1993, 363, 418-423);

[0422] (d) ribavirin (Van Voris, L. P. et al. “Antivirals for thechemoprophylaxis and treatment of influenza” Semin Respir Infect, 1992,7, 61-70);

[0423] (e) interferon (Came, P. E. et al. “Antiviral activity of aninterferon-inducing synthetic polymer” Proc Soc Exp Biol Med, 1969, 131,443-446; Gerone, P. J. et al. “Inhibition of respiratory virusinfections of mice with aeresols of synthetic double-strandedribonucleic acid” Infect Immun, 1971, 3, 323-327; Takano, K. et al.“Passive interferon protection in mouse influenza” J Infect Dis, 1991,164, 969-972);

[0424] (f) inactivated influenza A and B virus vaccines (“Clinicalstudies on influenza vaccine -1978” Rev Infect Dis, 1983, 5, 721-764;Galasso, G. T. et al. “Clinical studies on influenza vaccine-1976” JInfect Dis, 1977, 136 (suppl), S341-S746; Jennings, R. et al. “Responsesof volunteers to inactivated influenza virus vaccines” J Hyg, 1981, 86,1-16; Kilbourne, E. D. “Inactivated influenza vaccine” In: Plothin S A,Mortimer E A, eds. Vaccines Philadelphia: Saunders, 1988, 420-434;Meyer, H. M., Jr. et al. “Review of existion vaccines for influenza” AmJ Clin Pathol, 1978, 70, 146-152; “Mortality and Morbidity WeeklyReport. Prevention and control of Influenza: Part I, Vaccines.Recommendations of the Advisory Committee on Immunication Practices(ACIP)” MMWR, 1993, 42 (RR-6), 1-14; Palache, A. M. et al. “Antibodyresponse after influenza immunization with various vaccine doses: Adouble-blind, placebo-controlled, multi-centre, dose-response study inelderly nursing-home residents and young volunteers” Vaccine, 1993,11,3-9; Potter, C. W. “Inactivated influenza virus vaccine” In: Beare AS, ed. Basic and applied influeza research, Boca Raton, Fla.: CRC Press,1982, 119-158).

[0425] VIII. Therapies for the Treatment of Paramyxoviridae Infection

[0426] It has been recognized that drug-resistant variants of RSV canemerge after prolonged treatment with an antiviral agent. Drugresistance most typically occurs by mutation of a gene that encodes foran enzyme used in the viral replication cycle. It has been demonstratedthat the efficacy of a drug against RSV infection can be prolonged,augmented, or restored by administering the compound in combination oralternation with a second, and perhaps third, antiviral compound thatinduces a different mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution or other parameterof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous stresses on the virus.

[0427] Examples of agents that have been identified as active againstRSV, and thus can be used in combination or alternation with one or morenucleosides of general formula (I)-(XXIII) include:

[0428] (a) ribavirin (Hruska, J. F. et al. “In vivo inhibition ofrespiratory syncytial virus by ribavirin” Antimicrob Agents Chemother,1982, 21, 125-130); and

[0429] (b) purified human intravenous IgG-IVIG (Prince, G. A. et al.“Effectiveness of topically administered neutralizing antibodies inexperimental immunotherapy of respiratory syncytial virus infection incotton rats” J Virol, 1987, 61, 1851-1954; Prince, G. A. et al.“Immunoprophylaxis and immunotherapy of respiratory syncytial virusinfection in cotton rats” Infect Immun, 1982, 42, 81-87).

[0430] IX. Therapies for the Treatment of Abnormal CellularProliferation

[0431] Examples of agents that have been identified as active againstabnormal cellular proliferation, and thus can be used in combination oralternation with one or more nucleosides of general formula (I)-(XXIII)include:

[0432] A. Alkylating Agents

[0433] Nitrogen Mustards: Mechlorethamine (Hodgkin's disease,non-Hodgkin's lymphomas), Cyclophosphamide, Ifosfamide (acute andchronic lymphocytic leukemias, Hodgkin's disease, non-Hodgkin'slymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung, Wilms'tumor, cervix, testis, soft-tissue sarcomas), Melphalan (L-sarcolysin)(multiple myeloma, breast, ovary), Chlorambucil (chronic lymphocticleukemia, primary macroglobulinemia, Hodgkin's disease, non-Hodgkin'slymphomas).

[0434] Ethylenimines and Methylmelamines: Hexamethylmelamine (ovary),Thiotepa (bladder, breast, ovary).

[0435] Alkyl Sulfonates: Busulfan (chronic granuloytic leukemia).

[0436] Nitrosoureas: Carmustine (BCNU) (Hodgkin's disease, non-Hodgkin'slymphomas, primary brain tumors, multiple myeloma, malignant melanoma),Lomustine (CCNU) (Hodgkin's disease, non-Hodgkin's lymphomas, primarybrain tumors, small-cell lung), Semustine (methyl-CCNU) (primary braintumors, stomach, colon), Streptozocin (STR) (malignant pancreaticinsulinoma, malignant carcinoin).

[0437] Triazenes: Dacarbazine (DTIC;dimethyltriazenoimidazole-carboxamide) (malignant melanoma, Hodgkin'sdisease, soft-tissue sarcomas).

[0438] B. Antimetabolites

[0439] Folic Acid Analogs: Methotrexate (amethopterin) (acutelymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast, headand neck, lung, osteogenic sarcoma).

[0440] Pyrimidine Analogs: Fluorouracil (5-fluorouracil; 5-FU)Floxuridine (fluorodeoxyuridine; FUdR) (breast, colon, stomach,pancreas, ovary, head and neck, urinary bladder, premalignant skinlesions) (topical), Cytarabine (cytosine arabinoside) (acutegranulocytic and acute lymphocytic leukemias).

[0441] Purine Analogs and Related Inhibitors: Mercaptopurine(6-mercaptopurine; 6-MP) (acute lymphocytic, acute granulocytic andchronic granulocytic leukemia), Thioguanine (6-thioguanine: TG) (acutegranulocytic, acute lymphocytic and chronic granulocytic leukemia),Pentostatin (2′-deoxycyoformycin) (hairy cell leukemia, mycosisfungoides, chronic lymphocytic leukemia).

[0442] Vinca Alkaloids: Vinblastine (VLB) (Hodgkin's disease,non-Hodgkin's lymphomas, breast, testis), Vincristine (acute lymphocyticleukemia, neuroblastoma, Wilms' tumor, rhabdomyosarcoma, Hodgkin'sdisease, non-Hodgkin's lymphomas, small-cell lung).

[0443] Epipodophylotoxins: Etoposide (testis, small-cell lung and otherlung, breast, Hodgkin's disease, non-Hodgkin's lymphomas, acutegranulocytic leukemia, Kaposi's sarcoma), Teniposide (testis, small-celllung and other lung, breast, Hodgkin's disease, non-Hodgkin's lymphomas,acute granulocytic leukemia, Kaposi's sarcoma).

[0444] C. Natural Products

[0445] Antibiotics: Dactinomycin (actinonmycin D) (choriocarcinoma,Wilms' tumor rhabdomyosarcoma, testis, Kaposi's sarcoma), Daunorubicin(daunomycin; rubidomycin) (acute granulocytic and acute lymphocyticleukemias), Doxorubicin (soft tissue, osteogenic, and other sarcomas;Hodgkin's disease, non-Hodgkin's lymphomas, acute leukemias, breast,genitourinary thyroid, lung, stomach, neuroblastoma), Bleomycin (testis,head and neck, skin and esophagus lung, and genitourinary tract,Hodgkin's disease, non-Hodgkin's lymphomas), Plicamycin (mithramycin)(testis, malignant hypercalcema), Mitomycin (mitomycin C) (stomach,cervix, colon, breast, pancreas, bladder, head and neck).

[0446] Enzymes: L-Asparaginase (acute lymphocytic leukemia).

[0447] Biological Response Modifiers: Interferon-alfa (hairy cellleukemia, Kaposi's sarcoma, melanoma, carcinoid, renal cell, ovary,bladder, non Hodgkin's lymphomas, mycosis fungoides, multiple myeloma,chronic granulocytic leukemia).

[0448] D. Miscellaneous Agents

[0449] Platinum Coordination Complexes: Cisplatin (cis-DDP) Carboplatin(testis, ovary, bladder, head and neck, lung, thyroid, cervix,endometrium, neuroblastoma, osteogenic sarcoma).

[0450] Anthracenedione: Mixtozantrone (acute granulocytic leukemia,breast).

[0451] Substituted Urea: Hydroxyurea (chronic granulocytic leukemia,polycythemia vera, essential thrombocytosis, malignant melanoma).

[0452] Methylhydrazine Derivative: Procarbazine (N-methylhydrazine, M1H)(Hodgkin's disease).

[0453] Adrenocortical Suppressant: Mitotane (o,p′-DDD) (adrenal cortex),Aminoglutethimide (breast).

[0454] Adrenorticosteriods: Prednisone (acute and chronic lymphocyticleukemias, non-Hodgkin's lymphomas, Hodgkin's disease, breast).

[0455] Progestins: Hydroxprogesterone caproate, Medroxyprogesteroneacetate, Megestrol acetate (endometrium, breast).

[0456] E. Antioangiogenesis Agents

[0457] Angiostatin, Endostatin.

[0458] F. Hormones and Antagonists

[0459] Estrogens: Diethylstibestrol Ethinyl estradiol (breast, prostate)

[0460] Antiestrogen: Tamoxifen (breast).

[0461] Androgens: Testosterone propionate Fluxomyesterone (breast).

[0462] Antiandrogen: Flutamide (prostate).

[0463] Gonadotropin-Releasing Hormone Analog: Leuprolide (prostate).

[0464] X. Synthetic Protocol

[0465] Compounds of formula (I)-(XXIII) can be synthesized by any meansknown in the art. In particular, the compounds can be made via threedistinct routes: (a) from a pre-formed nucleoside, (b) condensation of amodified sugar or unmodified ribose with purine or pyrimidine, and (c)combination of the two routes. Since the 3-deoxy-D-erythropentofuranosestructure is found in the nucleoside antibiotic, cordycepin, a number oftotal syntheses of this antibiotic have been reported during 1960s (see:Lee, W. W. et al. J. Am. Chem. Soc., 1961, 83, 1906; Walton, E. et al.J. Am. Chem. Soc., 1964, 86, 2952; Suhadolnik, R. J. et al. Carbohydr.Res., 1968, 61, 545; Ikehara, M. et al. Chem. Pharm. Bull., 1967, 15,94; Kaneko, M. et al. Chem. Pharm. Bull. 1972, 20, 63). In a preferredembodiment of the invention, preparation of 3′-deoxy nucleosides frompreformed nucleosides are performed in the following ways;

[0466] A. Compounds of Types Ia-c and IIIa-c.

[0467] (i) Synthesis from Pre-Formed Nucleosides:

[0468] From the teachings of Marumoto, R. et al. Chem. Pharm. Bull.1974, 22, 128 where N4-acetylcytidine is treated with acetyl bromide togive 2′,5′-di-O-acetyl-3′-bromo-3′-deoxy-β-D-xylofuranosyl-cytosine (2,R=Ac), N⁴-protected-cytidine nucleosides can be derivatized to formpyrimidine nucleosides (I-a) as shown in Scheme 1.

[0469] An N⁴-protected-D-cytidine nucleoside 1 can be treated with anacid halide, such as acetyl bromide, to give the corresponding3′-halo-xylo-nucleoside 2. Deacetylation of 2 to 3, followed reductivedehalogenation affords the desired 3′-deoxycytidine derivatives 4.Treatment of 2 with an acid, preferably boiling aqueous acetic acid,gives the corresponding protected uracil nucleoside 5, which can bereadily converted into free 3′-bromo-xylo nucleoside 6a, from which3′-deoxyuridine derivatives 6b can be obtained by reductivedebromination. In a similar manner, starting fromN⁴-protected-L-cytidine, the L-enantiomer (III-a) of 4 and 6 can besynthesized.

[0470] In an alternate embodiment for the preparation of nucleosidesI-a, 2′, 5′-di-O-tritylation of a ribonucleoside gives 7 (R^(2′)R^(5′)=Tr) which is converted into the corresponding 3′-O-mesylates 8(Scheme 2). Treatment of 8 with diluted potassium or sodium hydroxidegives the corresponding xylo derivative 10 via anhydronucleoside 9,which, after de-O-tritylation, affords 12. Mesylation of 10, followed byde-O-tritylation yields the 3′-O-mesyl xylo-nucleoside. Upon treatmentof 8 with lithium bromide or sodium iodide, the corresponding3′-deoxy-3′-halogeno derivative 11 is formed via 9, which, afterde-O-tritylation, followed by hydrogenolysis, is converted into thedesired 3′-deoxyuridine derivative 6b. In a similar manner, startingfrom an L-ribonucleoside, the L-nucleoside (III-a) counterparts of 4 and6 are synthesized.

[0471] An example for the preparation of type I-b compound, purinenucleoside, is the synthesis of 3′-deoxypurine nucleosides (Scheme 3).Ribonucleoside 13 is treated with 2-methoxyisobutyryl halide (X=Cl orBr) to give a mixture of 3′-halogeno-xylo-furanosyl and 2′-halogeno-arabinofuranosyl derivatives (14 and 15). Hydrogenolysis,followed by chromatographic separation affords the corresponding3′-deoxynucleoside 17 along with the 2′-deoxynucleoside 16.Saponification of 17 gives the desired 3′-deoxynucleoside 20. Treatmentof the reaction mixture of 14 and 15 with a base gives the singleepoxide 18 in quantitative yield, which, upon treatment with ammonium orsodium iodide affords exclusively the 3′-xylo-iodide 19. Hydrogenolysisof 19 affords 20. Reduction of 18 with a reducing agent such as Raneynickel, lithium aluminum hydride or sodium borohydride also yields 20.

[0472] In a similar manner, starting from a purine L-ribonucleoside, theL-nucleoside counterpart of 20, which belongs to III-b, can besynthesized.

[0473] For the synthesis of a compound of formula I-c, the startingmaterial is a 5-nitropyrimidine or pyridine nucleoside (Scheme 4).Treatment of 5-nitrouridine (21, vide supra) with azide ion in a solventsuch as alcohol or dimethylformamide at a temperature range of from 20°C. to 100° C., preferably from 25° C. to 80° C. Nucleophilic attack ofazide ion at C-6 of 21 results in the formation of aci-nitro salt 22which cyclizes to 23. Neutralization of 23 furnishes the bicyclicnucleoside 24.

[0474] (ii) Synthesis by Condensation of an Appropriate Sugar with Base.

[0475] The appropriate sugar derivatives must be prepared forcondensation with the selected base. Though there are several methodsfor the synthesis of 3-deoxy-D-erythropentofuranose(3-deoxy-D-ribofuranose) derivatives (see: Lee, W. W. et al. J. Am.Chem. Soc., 1961, 83, 1906; Walton, E. et al. J. Am. Chem. Soc., 1964,86, 2952; Lin, T. -S. et al. J. Med. Chem., 1991, 34, 693; Ozols, A. M.et al. Synthesis, 1980, 557), new methods were developed for the presentinvention as shown in Scheme 5.

[0476] 1,2-O-Isopropylidene-5-O-methoxycarbonyl-cc-D-xylo-furanose (25)is converted into the corresponding 3-thiocarbonyl derivative26,followed by free radical deoxygenation using trialkyltin hydride in thepresence of a radical initiator, such as 2,2′-azobisisobutyronitrile.The deoxygenated product 27 is acylated with a mixture of acetic acid,acetic anhydride and sulfuric acid to give 28, which then is condensedwith a silylated base using Vorbruggen's procedure (see: Niedballa, U.et al. J. Org. Chem., 1976, 41, 2084; Vorbruggen, H. et al. Chem. Ber.,1981, 114, 1234; Kazinierczuk, Z. et al. J. Am. Chem. Soc., 1984, 106,6379) to obtain the pyrimidine nucleoside 29 (Type I-a) or a relatedpurine nucleoside (Type I-b). The 5-OH group can be alternativelyprotected with other acyl groups, such as benzoyls, p-nitrobenzoyls,p-chlorobenzoyls or p-methoxybenzoyls as well as other silyl groups,such as t-butyldimethylsilyl or t-butyldiphenyl groups. Similarly,L-xylose can be converted into the L-sugar counterpart of 25, which canbe further derivatized to attain the L-nucleoside of 30.

[0477] Alternatively, as shown in Scheme 6,1,2-O-isopropylidene-5-O-(t-butyldiphenylsilyl)-α-D-xylofuranose (31)can be sulfonylated with mesyl chloride, tosyl chloride or tresylchloride in pyridine to obtain32. After methanolysis of 32, the methylxyloside 33 can be treated with a base, such as sodium methoxide inmethanol, to afford the ribo-epoxide 34. Opening of the epoxide 34 withlithium aluminum hydride stereoselectively produces 3-deoxy sugar 36.Treatment of 34 with lithium bromide or sodium iodide in acetone or2-butanone gives 3-halogeno-3-deoxy xyloside 35. Reductivedehalogenation of 35 affords 36. Removal of the 5′-silyl protectinggroup with a fluoride ion source, such as tri-n-butylammonium fluoridein tetrahydrofuran or triethylammonium hydrogen fluoride gives 37.Acylation of 37 with acetic anhydride and acetic acid in the presence ofsulfuric acid gives tri-O-acetyl-3-deoxy-D-ribofuranose 38. Also,fluoride treatment converts 33 into 39, which, upon acetylation, affords40. These acetylated sugars 38 and 40 can be condensed withpertrimethylsilylated pyrimidine or purine bases using Vorbrueggen'sprocedure to give the 3′-modified nucleoside. The t-butyldiphenylsilylprotecting group can be replaced by t-butyldimethylsilyl group.

[0478] (iii) Post Synthetic Modifications (1-6)

[0479] (a) Modification at C-4 of Pyrimidine Nucleosides (I-a and III-a)

[0480] After condensation of 28 or 38 with uracil or 5-substituteduracil, the protected 3′-deoxyuridine derivative (29, R^(5′)═CH₃OCO,R^(2′)═Ac or R^(5′)═R^(2′)═Ac) is treated with phosphorus pentasulfidein pyridine or Lawesson's reagent in toluene to give 4-thiouracilnucleoside 41, which, upon treatment with ammonia, is converted into3′-deoxycytidine (43, R₁═R₂═H). Alternatively, methylation of 41 withmethyliodide or dimethylsulfate in base gives the 4-S-methyl derivative42. Displacement of the 4-S-methylgroup of 42 with various nucleophilesaffords the corresponding N⁴-substituted 3′-deoxycytidines 43. Also, 29can be converted into the 4-(triazol-2-yl) derivative 44, which can bereacted with ammonia or various amines to give 43. Alternatively,treatment of 44 with various alcohols or phenols affords thecorresponding 4-O-substituted-3′-deoxyuridines.

[0481] Alternatively, a uracil nucleoside, such as a sugar-protecteduridine 45 (R═H) i converted into the 4-(methylimidazolium) 46 (Scheme8) or 4-O-(2,4,6-triisopropylbenzenesulfonyl) intermediate 47 and thentreated with a nucleophile, such as hydroxylamine, to give thecorresponding C-4 modified nucleoside, such as N4-hydroxy-X cytidine(48, R═H).

[0482] In similar manners starting from the L-nucleoside counterparts,the corresponding III-a nucleosides are prepared.

[0483] (b) Modification at C-5 of Pyrimidine Nucleosides (I-a and III-a)

[0484] (i) Halogenation (Scheme 9)

[0485] 3′-Deoxyuridine (6, R═H) can be fluorinated with fluorinatingagents, some non-limiting examples include fluorine in acetic acid,selectfluor in an inert solvent or solvents such as tetrahydrofuran orcesium fluoroxisulfate in alcohol (see: Stovber, S. et al. J. Chem. Soc.Chem. Commun., 1983, 563), to give 5-fluoro-3′-deoxyuridine (49). The5-chloro, 5-bromo and 5-iodouridine derivatives (50-52) are obtainedusing the appropriate N-halogenosuccinimde. Treatment of 6 with brominein water or iodine in acetic acid in the presence of an oxidizing agentsuch as nitric acid affords the 5-bromo- or 5-iodo-uracil nucleoside,respectively. The cytosine derivative 43 (R═H) also can be convertedinto the corresponding 5-halogeno derivative (44-56).5-Fluoro-3′-deoxycytidine (53, R═H) is prepared by condensing 28 or 38with 5-fluorocytosine, followed by saponification.

[0486] In similar manners starting from the L-nucleoside counterparts,the corresponding III-a nucleosides are prepared. Scheme 10 depicts theconversion of the brominated compound 51 into 5-hydroxy-3′-deoxyuridine(63) by treatment with sodium bicarbonate solution. Alkylation of 55with an alkyl iodide with base affords 62. Prolonged reaction of 51 withan alkali metal cyanide gives the 5-cyano-uracil derivative 57, whichcan be hydrated to 5-carboxamide 58 and 5-carboxylic acid 59. Conversionof 59 into an alkyl ester 60, followed by reduction with sodiumborohydride yields the 5-hydroxymethyl derivative 61. Compound 60alternatively can be treated with dihydropyran and a catalytic amount ofacid, such as hydrochloric, sulfuric or p-toluenesulfonic acid, to yieldthe 2′,5′-di-O-protected nucleoside 64. Sodium borohydride reduction of64 affords 65. Due to allylic nature of 65, treatment with mesylchloride or tosyl chloride gives the 5-chloromethyl-uracil derivative66. Alkoxide treatment of 66, followed by deprotection gives thecorresponding 5-alkoxymethyl-3′-deoxyuridine (69). Similarly, reactionof various amines with 66 affords 67, which, upon mild acid hydrolysis,is converted into 68. Reaction with 66 and thiourea gives mercaptomethylderivative (70, R═H), while treatment with sodium mercaptide givesthioalkyl derivative 70 (R=alkyl), which can be oxidized with hydrogenperoxide to the corresponding sulfone (71).In similar manners startingfrom the L-nucleoside counterparts, the corresponding III-a nucleosidesare prepared.

[0487] (ii) Nitration (Scheme 11)

[0488] Treatment of uridine 6 with nitronium tetrafluoroborate insulfolane (see: Huang, G. -F. et al. J. Org. Chem., 1977, 42, 3821;Huang, G. -F. et al. J. Carbohyd. Nucleosides Nucleotides, 1978, 5, 317)affords the corresponding 5-nitro derivative 72. Catalytic hydrogenationof the nitro-nucleoside 72 gives the corresponding 5-amino derivative73. Diazotization of 73 with nitrous acid gives the5-diazo-3′-deoxyuridine (74), which, upon hydrolysis, can be convertedinto the 1,2,3-triazole 75. Similar conversions of 5-aminouridine intoribosilyltriazole have been reported (see: Roberts, M. et al. J. Am.Chem. Soc., 1952, 74, 668; Thurber, T. C. et al. J. Am. Chem. Soc.,1973, 95, 3081; J. Org. Chem., 1976, 41, 1041). Reaction of 72 withsodium azide in dimethylformamide affords the triazolopyrimidine(8-azapurine) nucleoside 76.

[0489] In similar manners starting from the L-nucleoside counterparts,the corresponding III-a nucleosides are prepared.

[0490] A similar sequence of reactions is shown in Scheme 12, startingfrom 3′-deoxycytidine 4 gives 5-nitro-3′-deoxycytidine (77), followed by5-amino-3′-deoxycytidine (78). However, treatment of 78 with nitrousacid results in the formation of another 8-azapurine nucleoside 79. Thesame sequence of reactions can be applied to the correspondingL-nucleosides III-a.

[0491] (iii) Hydroxymethylation

[0492] Treatment of 6 (R═H, R^(5′)═R^(3′)═R^(3″)H) with formaldehyde inbase such as aqueous potassium hydroxide or sodium hydroxide gives5-hydroxymethyl-3′-deoxyuridine (80) as shown in Scheme 13, which isconverted into 5-ethoxymethyl-3′-deoxyuridine (81, X═OCH₂CH₃) bytreatment with ethanolic hydrogen chloride. Compound 80(R^(5′)=R^(3′)═TBDPS) can also be prepared from the thymine derivative 6(R═CH₃, R⁵═R³═TBDPS) by photochemical bromination to 81 (X═Br), followedby hydrolysis (Matulic-Adamic, J. et al. Chem. Pharm. Bull., 1988, 36,1554). Compound 80 is converted into 5-chloromethyl derivative (81,X═Cl) by action of hydrochloric acid or 5-fluoromethyl derivative (81,X═F) by treatment with diethylaminosulfur trifluoride (DAST). Oxidationof 80 (R^(5′)═R²═TBDPS, R^(3″)═H) with manganese dioxide affords the5-formyl derivative 82, which is a good substrate for various reactionsincluding Wittig, Wittig-Horner, Grignard or Reformatsky reaction. Forexample, treatment of 82 with ethoxymethylene triphenylphosphorane[EtOC(═O)CH═PPh₃] gives 5-(2-ethoxycarbonyl)ethylene-3′-deoxyuridinederivative (83), which can be converted into 5-ethylene-,5-(2-chloroethylene)- or 5-(2-bromoethylene)-3′-deoxyuridine derivative(85) by way of the 5-(ethylene-2-carboxylic acid) derivative 84.5-Difluoromethyl derivative 86 can be obtained by treatment of 82 withDAST. These synthetic pathways are shown in Scheme 13.

[0493] The same sequence of reactions can be applied to thecorresponding L-nucleosides III-a.

[0494] (iv) Metallation

[0495] In aqueous buffer, 6 or 4 can be treated with mercuric acetate,followed by sodium chloride, to give the corresponding 5-chloromercuriderivative 87 or 91, respectively (Scheme 14), in quantitative yield.Reaction of 87 or 91 with iodine in ethanol gives the 5-iodo derivative52 or 56, respectively. Compound 52 can be converted to 5-ethynylderivatives 88 by reaction with 1-alkynes andbis(triphenylphosphine)palladium chloride (Ph₃P)₂PdCl₂ in the presenceof cuprous iodide and triethylamine. Treatment with trifluoroiodomethaneand powdered copper, on the other hand, converts 52 into5-trifluoromethyl-3′-deoxyuridine 89. Treatment of 87 with lithiumpaladium chloride (Li₂PdCl₄) and alyl chloride affords5-allyl-3′-deoxyuridine (90). Methyl acrylate reacts with 87 or 91 inthe presence of Li₂PdCl₂ to give5-(E)-(2-methoxy-carbonyl)vinyl-3′-deoxyuridine (83) or -cytidine (92),respectively. Saponification of 83 to 84, followed byN-halogenosuccinimide yields 5-(E)-halogenovinyluracil nucleoside 85(X═Cl, Br or J). Thermal decarboxylation of 84 gives 5-vinyluracilderivative 85 (X=H). Compound 85 (X═H) c an also be prepared bytreatment of 52 with vinyl acetate in the presence of palladiumacetate-triphenylphosphine complex. Similarly, 91 can be converted intothe corresponding acrylate derivative 92, which, after hydrolysis to 93,is reacted with N-halogenosuccinimide to give5-(E)-(2-halogenovinyl)-3′-deoxycytidines (94). It should be noted thatcatalytic hydrogenation of 5-vinyl derivatives gives the corresponding5ethyl-pyrimidine nucleosides. Hydration of 5-ethynyl-3′-deoxyuridine(88, R═H) with diluted sulfuric acid gives 5-acetyl-3′-deoxyuridine inhigh yield.

[0496] In a similar manner but starting from the L-nucleosidecounterparts, the corresponding III-a nucleosides are prepared.

[0497] (c) Modification at C-6 of Pyrimidine Nucleosides (I-a and III-a)

[0498] Treatment of 5-bromo-3′-deoxyuridine (51, Scheme 15) with sodiumor potassium cyanide in dimethylformamide at room temperature affords6-cyano-3′-deoxyuridine (95) in high yield. Further treatment atelevated temperature converts 95 into the 5-cyano isomer 59. Hydrolysisof 95 furnishes 3′-deoxyorotidine 96. Methanolysis of 95 gives themethyl ester 97, which, upon amminolysis, is converted into 98, whereinR′ is lower alkyl of from C₁ to C₆ or benzyl or phenyl group. Reductionof 97 with sodium borohydride affords 6-hydroxymethyl derivative 99,which is converted into 6-chloromethyluracil nucleoside 100 by action ofhydrochloric acid. Reaction with various amines, 100 is converted intothe corresponding 6-aminomethyl-3′-deoxyuridine (101). A similarsequence of reactions starting from 3′-deoxycytidine (55) gives3′-deoxycytidin-6-yl-carboxylic acid (103) or its methyl ester 104 viathe 6-cyano intermediate 102. Various 6-carbox-amidocytosine nucleosides105 can be obtained by treatment of 104 with the corresponding amines.Borohydride reduction of 104 affords 6-hydroxymethyl derivative 104which can be converted into 6-chloromethyl-3′-deoxycytidine 107 byaction of hydrochloric acid. Compound 107 can be converted into thecorresponding 6-aminomethyl-3′-deoxcytidine (108) by reaction withvarious amines. The same sequence of reactions can be applied to thecorresponding L-nucleosides III-a.

[0499] Further derivatization is shown in Scheme 16. Lithiation ofuracil and cytosine nucleosides occurs at C-6.(see: Tanaka, H. et al.Tetrahedron Lett., 1979, 4755; Sergueeva, Z. A. et al. NucleosidesNucleotides Nucleic Acids, 2000, 19, 275) Thus, treatment of fullytrimethylsilylated 3′-deoxycytidine (109, R^(3′)═R^(3″)═H) withn-butyllithium at −45° C., followed by treatment with methyl iodide orcarbon dioxide, gives 6-methyl-3′-deoxycytidine (110) or3′-deoxycytidine-6-carboxylic acid (103), respectively. In a similarmanner but starting from the L-nucleoside counterparts, thecorresponding III-a nucleosides are prepared.

[0500] Treatment of 2′,5′-di-O-(tetrahydropyran-2-yl)-3′-deoxyuridine(6, R^(2′)═R^(5′)═THP, R^(3′)═R^(3″)═H) with lithium diusopropylamide intetrahydrofuran at −78° C. and subsequent reaction with alkyl halideresult in the formation of 6-alkyl-3′-deoxyuridines (111). Oxidation of111 (n=0) with selenium dioxide gives 3′-deoxyuridine-6-carboxaldehyde(112), which, upon treatment with nitromethane in the presence of basegives the nitroalkene 113. Compound 112 reacts with various Wittigreagents to give the corresponding olefins 114-117. Also, Grignardtreatment of 112 gives 6-hydroxyalkyl derivatives 121. Oxidation of 121affords the corresponding 6-acyl derivatives 120 (R=alkyl). On the otherhand lithiated 6 (R^(5′)═R^(2′)═THP, R^(3′)═R^(3″)′H) with benzaldehydeproduces 6-hydroxybenzyl derivative 119 which is converted into6-benzoyl-3′-deoxyuridine (120, R═Ph) by mild oxidation. Also, reactionof lithiated 6 with diphenyldisulfide affords6-phenylthio-3′-deoxyuridine 118, as shown in Scheme 17.

[0501] In a similar manner but starting from the L-nucleosidecounterparts, the corresponding III-a nucleosides are prepared.

[0502] (d) Modification at C-6 of Purine Nucleosides (I-b and III-b)

[0503] Compound 28 or 38 is converted into halogenase 122 (Scheme 18) bytreatment with hydrogen chloride or hydrogen bromide in acetic acid orhydrogen bromide in dichloromethane and condensed with 6-chloropurine bythe sodio procedure in acetonitrile affords 3′-deoxynucleoside 123.Aqueous sodium or potassium hydroxide treatment of 123 gives3′-deoxyinosine (124). Treatment of 123 with sodium methoxide inmethanol affords 6-O-methyl-3′-deoxyinosine (125). Mild saponification,followed by catalytic 110 hydrogenolysis of 123 results in theproduction of 3′-deoxynebularine (126). Thiourea reacts with 123 to give6-thiopurine nucleoside 127, which is S-alkylated to 128. Compounds 123,127 and 128 readily react with various amines, hydroxylamine, hydrazineand aminoalcohols to give 3′-deoxyadenosine analogues 129-133. Treatmentof 123 with sodium azide gives 6-azidopurine nucleoside 134.

[0504] The same sequence of reactions can be applied to thecorresponding L-nucleosides III-b.

[0505] These compounds can also be synthesized by nitrous acid treatmentof 6-hydrazidopurine nucleoside 130. Reduction of 129, 130 or 134 gives3′-deoxyadenosine (i.e., cordycepin). Compound 125 or cordycepin areexpected to be converted in vivo into 124 by action of adenosinedeaminase. 6-Unsubstituted purine nucleoside 126 may be oxidized in vivoto 124.

[0506] Condensation of 122 with 2-substituted-6-chloropurine gives the2-substituted analogue of 123. The 6-chloro functionality can beconverted into various functional groups by nucleophilic substitutionreactions. Thus, 2-amino-6-chloropurine is converted into 135 (Scheme19), which can be converted various 2-aminopurine nucleosides (136-147).It should be noted that the 2,6-diamino-(141) and 2-amino-purine (138)nucleosides are potential precursors for 3′-deoxy-guanosine (136). In asimilar manner but starting from the L-nucleoside counterparts, thecorresponding 111-b nucleosides are prepared.

[0507] In a similar manner, 2-oxo-, 2-methoxy-, 2-thio-,2-alkylmercapto-, 2-methyl-, 2-methyl-amino- or 2-dimethylamino-purinenucleosides (148-154) are synthesized (Scheme 20). Also, in a similarmanner but using the corresponding L-nucleosides, compounds of III-btype are prepared.

[0508] (e) Modification at C-2 of Purine Nucleosides (I-b and III-b).)

[0509] The 2-amino group of 135-147 can be modified to obtain 155(Scheme 21) by acylation with various alkanoyl or aroyl halides. Then,155 can further be derivatized into the corresponding 2-alkylamino or2-arylamino derivative 156 by reduction with a borane-amine complex(Sergueeva, Z. A. et al. Nucleosides Nucleotides Nucleic Acids, 2000,19, 275). Alternatively, the 2-amino group of compound 135 can besubstituted by undergoing a Schiemann reaction, diazotizing in thepresence of fluoroborate, followed by thermal decomposition, to give2-fluoro-6-chloropurine nucleoside 157. Furthermore, the 6-chlorosubstituent of these nucleosides can be displaced with variousnucleophilic reagents as described above. It should be noted that thepresence of 2-fluoro substituent protects the 6-amino group fromadenosine deaminase attack.

[0510] (f) Modification at C-8 of Purine Nucleosides (I-b)

[0511] It should be noted that modification of the 8-position of purinenucleosides is important as the substitution at this position alters thepreferred conformation of the nucleosides to be syn.

[0512] Cordycepin (158, R^(3′)═R^(3″)═H), 3′-deoxyinosine (124,R³═R^(3″)═H) and 3′-deoxyguanosine (136, R^(3′)=R^(3″)=H) can bebrominated at the C-8 position by treatment with bromine in acetic acidin the presence of sodium acetate to 159-161 (Scheme 22). The C-8bromine substituent in 159-161can be replaced with sulfur by the actionof thiourea to obtain 162-164, which can be alkylated or aralkylatedwith alkyl or aralkyl halide in a polar solvent, such as water, alcoholor dimethylformamide, in the presence of base, such as sodium orpotassium carbonate, to give 165-167. The methylmercapto derivative165-167 (R=methyl) can be oxidized to the corresponding sulfone 168-170.Upon treatment of these sulfones with various amines, the corresponding8-amino derivatives 171-173 are obtained. Many of the 8-aminoderivatives can be obtained directly from 159-161 by treatment withamines. Also, 159 can be converted into the 8-oxo derivative 174 bytreatment with sodium acetate in acetic anhydride, followed byhydrolysis. O-Alkylation of 174 with triethyloxonium fluoroborate gives8-ethoxycordycepin 175.

[0513] 8-Alkyl derivatives 176 (Scheme 23) are prepared from 123(R^(5′)=R^(2′)=THP) by treatment with lithium diisopropylamide intetrahydrofuran below −70° C., followed by alkyl halide treatment. Thismethod was successfully used in other ribonucleosides (Tanaka, H. et al.Chem. Pharm. Bull., 1983, 31, 787) but never been applied to3′-deoxynucleosides. When carbon dioxide is used instead of alkylhalide, purine nucleo side 8-carboxylic acid 177 is obtained.Esterification to 178, followed by ammonolysis gives amide 181, which isdehydrated to 8-cyanopurine nucleoside 182. Reduction of 178 withborane-dimethylsulfide affords the alcohol 179. Mild oxidation withdimethylsulfoxide and oxalic chloride affords aldehyde 180. Compounds179 and 180 are versatile intermediates for various modifications.

[0514] B. Compounds of Types IIa-c and IVa-c.

[0515] (i) Synthesis from Pre-Formed Nucleosides:

[0516] Several methods are available to introduce a 2′,3′-unsaturationinto a preformed nucleosides. An example is shown in Scheme 24.

[0517] Selective O-silylation of nucleoside 7, preferably witht-butyldimethylsilyl halide or t-butyldiphenylsilyl halide, in base,preferably in pyridine at from 0° C. to 80° C., preferably at roomtemperature, followed by sulfonylation, preferably with mesyl chlorideor tosyl chloride in base, preferably in pyridine at from 0° C. to 80°C., preferably at room temperature, gives 8 in high yield, which can bereadily converted into the lyxo-epoxide 183 by treatment with base.Reaction of 183 with halide ion, preferably iodide ion, such astreatment with sodium iodide in acetone or methylethylketone givesexclusively the trans-iodohydrin 184, X =1). Mesylation of 184 gives inhigh yield of the olifin 186 via 185. Compound 185 can be isolated inpoor yield after short reaction time. De-O-silyation of 186 withfluoride, such as tetrabutyl ammonium fluoride affords the desiredolefin 187, type II-a nucleoside, in high yield.

[0518] Starting from 2′-deoxy nucleosides, e.g., 188 (Scheme 25), thetype II-a olefinic sugar nucleoside also can be prepared. Sulfonylationof 188, preferably with mesyl chloride in pyridine at temperature rangefrom −10° C. to 80° C., preferably at room temperature, gives thedi-O-mesylate 189, which, upon treatment with aqueous base such assodium hydroxide solution gives 3′,5′-anhydrosugar nucleoside 190. Thelatter nucleoside can be readily converted into the desired 187 in highyield by treatment with strong, anhydrous base, such as with potassiumtert-butoxide in dimethylsulfoxide at temperature range of from −10° C.to 80° C., preferably at room temperature for 10 minutes to overnight,preferably 1.5 to 3 hours.

[0519] An example for preparation of 2′-substituted olefinic sugarnucleoside of type II-a is given in Scheme 26.1-(2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl)thymine (191) is selectivelyprotected, preferably with trityl chloride or t-butyldimethylsilylchloride or t-butyldiphenylsilyl chloride, in pyridine to give 192.Sulfonylation of 192, preferably with mesyl chloride in pyridine, givesthe mesylate 193, which, upon treatment with non-nucleophilic base, suchas DBU or DBN in anhydrous inert solvent, such as methylene chloride,affords 2,3′-anhydro nucleoside 194. This compound is readily convertedinto 2′-fluoro-olefinic sugar nucleoside 195 upon treatment withpotassium t-butoxide in dimethylsulfoxide. De-protection of 195 givesthe desired 2′-fluorinated II-a type nucleoside 196. 5′-O—Silylprotection gives better overall yield than trityl protection.

[0520] All these reactions can be applied to the correspondingpyrimidine L-nucleosides for the preparation of IV-a type nucleosides.

[0521] Nucleosides of type I-b can be prepared readily from 197 (Scheme27). Selective protection of 197 at the 5′-position, e.g., witht-butyldimethylsilyl or t-butyldiphenylsilyl group affords 198.Sulfonylation with tosyl halide or mesyl halide in base such as inpyridine affords the protected olefinic nucleoside 199. De-O-silylationof 199 with fluoride, such as tetrabutyl ammonium fluoride affords thedesired olefin 200, type I-b nucleoside, in high yield.

[0522] Alternatively, treatment of 15 (see Scheme 3) with chromousacetate gives, after deprotection with base 200 in good yield.

[0523] By the same procedure but using purine L-nucleosides, thecorresponding olefinic sugar L-nucleosides of type IV-b can be obtained.

[0524] (ii) Synthesis by Condensation of Base and Unsaturated SugarDerivative

[0525] Commercially available 4-hydroxymethyl-2-pentenone (201, Scheme28) is silylated, preferably with t-butyldimethylsilyl halide in base,preferably in pyridine, to give 202, which is reduced with borohydrideto 203. After acetylation, the product 204 is condensed with silylatedbase, e.g., 5-substituted uracil. A complicated mixture is 7ff obtainedin which the anomeric nucleosides (205) are the major components. Afterchromatographic separation of the anomers 206 and 207, followed bydesilylation of each anomer affords the P-nucleoside 208 (type II-a) anda-nucleoside 209 (type XVIII-c), respectively.

[0526] Another example is shown in Scheme 29. 2-Fluoro-lactone 212 canbe prepared by Wittig condensation of aldehyde 210 with Ph₃P═CFCO₂Et.Silyl protection and DIBAH reduction, followed by acetylation of theproduct affords 213. Condensation of 213 with silylated purine, such as6-chloropurine, in the presence of Lewis acid, such as trimethylsilyltriflate or tin tetrachloride, in an inert solvent, such as methylene orethylene chloride gives anomeric mixture 214. These anomers areseparated on a silica gel column. After desilylation of each component,the corresponding β-nucleoside 215 (type II-b) and (x-nucleoside 216(type XVIII-d) can be obtained.

[0527] C. Synthesis of Carba-Sugar Nucleosides (V-X)

[0528] Only carba-nucleosides so far found in nature are adeninenucleosides, i.e., aristeromycin and neplanocins, and they are eitherextremely expensive or commercially not available. Thus, these types ofnucleosides are chemically synthesized from scratch. The carba-sugarderivative is prepared first and then the heterocyclic aglycon is Itconstructed on the sugar to prepare carba-sugar nucleosides or in thecase of purine nucleoside, the base is directly condensed with thecarba-sugar.

[0529] Scheme 30 illustrates the synthesis of 5-fluoro-garba-cytidine(227, Type V-a). The carba-sugar interrediate 219 can be synthesized byany means known in the art. It is disclosed by Ali et al. (TetrahedronLetters, 1990, 31, 1509) that D-ribonolactone 217 is converted into thepentanone intermediate 218. The ketone 218 can then be reduced by anyknown reducing agent, preferably sodium borohydride in methanol at 0° C.for 1 hour to afford alcohol 219. Sulfonylation of 219, preferably withmesyl chloride in methylene chloride in the presence of triethylamine at0° C. for 2 hours gives 220, which is then treated with sodium azide inDMF at 140° C. ovemight to give 221. The azide 221 can readily bereduced with any known reducing agent, e.g., Ph₃P (Staudinger procedure)or catalytic hydrogenolysis, preferably over palladium on carbon. Theresulting amine 222 is subjected to Warrener-Shaw reaction withβ-methoxyacryloylisocyanate in DMF, followed by ammonium hydroxidetreatment to form protected carba-uridine 224 via the linearintermediate 223. Protected 5-fluoro-carba-uridine (225) can be obtainedby fluorination of 224 with any fluorinating agent. Preferably, thefluorinating agent is fluorine in acetic acid. After quenching thereaction with base, preferably triethylamine. Conversion of uracilnucleoside 225 into protected carba-5-fluorocytidine (226) can beachieved in a similar manner as described with Scheme 7. The protectinggroups of 226 are removed with acid, preferably with trifluoroaceticacid/water (2:1 v/v) at 50° C. for 3 hours, to give 227.

[0530] Sulfonylation of 219 with triflyl chloride in methylene chloridein the presence of triethylamine gives triflate, which, upon reactionwith purine base, such as adenine, and sodium hydride in an inertsolvent, such as acetonitrile or DMF directly affords the correspondingpurine nucleoside (V-b type).

[0531] By using the same procedure but starting from L-ribonolactone,the corresponding L-nucleosides counterparts (type VIII nucleosides) canbe obtained.

[0532] Alternatively, commercially available(1R)-(−)-azabicyclo[2.2.1]hept-5-en-3-one (228, Scheme 31) is convertedinto 2,3-dihydroxy-lactam 229 by osmium tetroxide oxidation. Aftermethanolysis of 229 with methanolic hydrogen chloride, the product 230is treated with 2,2-dimethoxypropane in acetone orl,l-dimethoxycyclohexane in cyclohexanol to give a ketal, e.g., 231,which is reduced to 232 with sodium borohydride. The aminoalcohol 232 isconverted into 2′,3′-O-cyclohexylidene-carba-uridine by reaction withβ-methoxyacryloylisocyanate, followed by ammonia treatment. Acidtreatment, preferably with trifluoroacetic acid in methanol, givescarba-uridine (233). carba-5-Fluorocytidine (227) can be obtainedreadily from 233 by the well-known means in the art.

[0533] In a similar sequence of reactions but starting from the otheroptical isomer, (IR)-(+)-azabicyclo[2.2.1]hept-5-en-3-one, thecorresponding L-nucleoside analogue (type VIII) can be obtained.

[0534] Nucleoside of type VI is prepared from nucleoside of type V. Anexample is shown in Scheme 32. Aristeromycin (234) or anycarba-ribonucleoside is converted into the correspondingN-[(dimethylamino)methylene]-5′-O-trityl derivative 235 by treatmentwith dimethylformamide dimethylacetal in DMF, followed by tritylation.Reaction of 235 with thiocarbonyldiimidazole gives 2′,3′-O-thiocarbonate236, which, upon radical reduction with tri-n-butyltin hydride in thepresence of 2,2′-azobis(2-methylpropionitrile) affords olefin 237 alongwith 3′-deoxy- and 2′-deoxy-aristeromycine derivatives 238 and 239,respectively. These products can be readily separated on a silica gelcolumn. Each of these produces the corresponding free nucleoside, 240,241 and 242, respectively, upon acid treatment. This procedure isparticularly suited for preparation of small amounts of severalnucleosides in short time for screening.

[0535] By the same procedure but using type VIII nucleosides instead oftype V, the corresponding L-nucleosides of type IX can be obtained.

[0536] Stereoselective conversion of type V to type VI is also possibleas shown in Scheme 33. 5-Fluoro-carba-uridine (233) is converted intothe 5′-O-trityl-2′,3′-di-O-mesylderivative 243. Aqueous base treatmentof 243 affords lyxo epoxide 245 via 2,2′anhydro nucleoside intermediate244. Epoxide ring-opening with sodium iodide in acetone or butanonegives trans iodohydrin 246, which, upon mesylation affords the olefin248 via 247. De-O-tritylation of 247 furnishes 249. Instead of5′-O-trityl protection, silyl protection with t-butyldimethylsilyl ort-butyldiphenylsilyl protection can also be used. Also, instead ofmesylation, other sulfonylation using an agent, such as tosyl chloride,trifyl chloride or triflyl anhydride can be used.

[0537] By using the same procedure but using type VIII nucleosidesinstead of type V, the corresponding L-nucleosides of type IX can beobtained.

[0538] Also, nucleosides of type VI-b can be synthesized starting from2-cyclopenten-1-one (250, Scheme 34). Michael addition oft-butoxymethyllithiumcuprate [(t-BuOCH₂)₂CuLi] to 250 yields the adduct251. Phenylselenation of 251 according to Wilson et al. (Synthesis,1995, 1465) mainly occurs trans to t-butoxymethyl group to give 252.DIBAH reduction reduces the carbonyl group to hydroxyl group in astereoselective manner to give 253. Sulfonylation, preferably withtriflyl chloride or triflic anhydride in base, to 254, followed bycondensation with sodio-purine, produced, e.g., adenine and NaH, in aninert solvent such as acetonitrile affords 255 in a stereoselectivemanner. Oxidation of the selenide 255 with hydrogen peroxide in pyridinesmoothly converts 255 into the olefin 256. Mild acid treatment of 256gives free nucleoside 240.

[0539] Alternatively, acetylation of 253, followed by condensation withsilylated pyrimidine, such as tris(trimethylsilyl)-5-fluorocytosine inthe presence of trimethylsilyl trifluoromethylsulfonate gives high yieldof the corresponding pyrimidine nucleoside, from which VI-a typenucleoside can readily prepared by oxidation and acid removal of t-butylgroup of the product.

[0540] By using the same procedure but using type VIII nucleosidesinstead of type V, the corresponding L-nucleosides of type IX can beobtained.

[0541] Furthermore, racemic carba analogues of 2′,3′-unsaturatednucleosides can be prepared by the procedure of Shi et al. (J. Med.Chem., 1999, 42, 859) who achieved multi-step preparation of racemiccis-3,4-epoxy-cyclopentanemethanol 257 (Scheme 35) from ethylcyclopentene-4-carboxylate. Opening of the epoxide withdiphenyldiselenide affords 258, which, after acetylation followed byperoxide treatment, gives diacetate 259. Treatment of 259 withsodiopyrimidine, prepared by reaction of uracil or cytosine derivativewith NaH in dimethylsulfoxide, in the presence of Pd(PPh₃)₄ in an inertsolvent, e.g., tetrahydrofuran, gives 260 in 10-70% yield afterdeacetylation of the product.

[0542] Scheme 36 shows the synthesis of 3,4-unsaturated carba nucleosideof type VII Wolfe et al (J. Org. Chem., 1990, 55, 4712) prepared 261from D-ribonolactone. Quenching the Michael addition of t-butoxyrnethylgroup to (261, Scheme 36) with sulfinyl chloride, followed by heatingthe product with calcium carbonate gives cyclopentenone 262. Reductionof 262 with DTBAH followed by sulfonylation affords 263. Condensation of263 (preferably R═CF₃) with purine base with NaH as described earliergives purine nucleoside VII-b, e.g., neplanocin A (264). Treatment of263 (preferably R═Me) with NaN₃ gives 265 which can be readily convertedinto various pyrimidine nucleosides (VII-a) including 266 by theprocedure already described with Scheme 30.

[0543] Starting from L-ribonolactone, the corresponding L-nucleosidecounterparts (X-a and X-b) can be readily prepared.

[0544] D. Synthesis of Nucleosides of Types XI and XII.

[0545] There are several methods are available for the synthesis ofthese types of nucleosides, Some nucleosides used in the presentinvention are prepared mainly in the following manner. 1-Mentylester of2,2-dimethoxyacetic acid (267, Scheme 37) is condensed with thioglycolicacid to give a diastereomeric mixture 268, which can readily beseparated on a silica gel column. Reduction of 268 with NaBH₄ inethanol, followed by acetylation affords 269, which is condensed withsilylated base in the presence of tin tetrachloride. Mainly the desiredprotected β-nucleoside is obtained and is purified by chromatography.De-O-acetylation affords the corresponding unprotected nucleoside 270.Also, 270 is obtained starting from 2,2-dimethoxyethyl ester ofN-t-Boc-L-proline. This compound is treated with 3 equivalents ofthioglycolic acid in methylene chloride in the presence of MgSO₄ and CASto give 271 as a diastereomeric mixture, which is separatedchromatographically. Reduction of each diastereomer of 271 withLi(t-BuO)₃AIH in tetrahydrofuran and subsequent acetylation affords 272,which is condensed with silylated base, followed by deprotection of theproduct to give 270.

[0546] Nucleosides of type XIII used in this invention are prepared byusing means known in the art. In a preferred embodiment, XIII-a typenucleosides are prepared in one or two-step synthesis reported (NucleicAcid Chem., 1978, 1, 272 and 343) by activating the 5′-OH bysulfonylation followed by base treatment or direct treatment ofunprotected nucleosides with Ph3P and diethyl diazocarboxylate.

[0547] Preparation of nucleosides of type XIV used in the presentinvention are synthesized by methods somewhat analogous to thoseutilized for the synthesis of the corresponding 5-fluorodeoxyuridineadducts by Duschinsky et al. (J. Med. Chem., 1967, 10, 47). Someexamples are shown in Scheme 38 using 5-fluorouridine (273). Anypyrimidine nucleoside containing a strongly electron-withdrawingsubstituent at C-5 undergoes similar adduct formation. Treatment of 273with bromine in methanol gives adduct 274 which can be reduced to 275 bycatalytic hydrogenation. Treatment in water gives the bromohydrin 277while action of bromine in acetic acid in the presence of aceticanhydride affords 276. The corresponding other adducts can be preparedby using other hypohalites, e.g., hypochlorite gives 278. Each of theseadducts are diastereomeric mixture and are used for screening as such.

[0548] E. Nucleosides of Type XV-XVIII.

[0549] Nucleosides used in this invention are prepared by oxidation of4-thiouridine and 6-thiolnosine derivatives according to the well-knownmeans in the art. Type XVI compounds are C-nucleosides. XVI-anucleosides are synthesized from ψ-uridine by methods known in the art(Watanabe, “The Chemistry of C—Nucleosides”, Townsend, L. 4 B., Ed., In“Chemistry of Nucleosides and Nucleotides”, Plenum, Publ., New York,Vol., 3, 421, 1994), or condensation of an aromatic compound toprotected ribonolactone, followed by manupulation of the products (e.g.,Kabat et al., J. Med. Chem., 1987, 30, 924). Nucleosides XVI-b and XVI-care prepared according to a modified procedure developed by Pankiewiczet al., (Carbohydr. Res., 1984, 127, 227; Nucleosides Nucleotides, 1991,10, 1333). The purine-type XVI-d C-nucleosides are synthesized accordingto the method reported by Chu et al., (J. Heterocycl. Chem., 1980, 17,1435). Nucleosides of type XVII used in this invention are synthesizedeither by cross-aldol reaction of 4′-formyl nucleosides withformaldehyde or condensation of preformed sugar with a base. Preparationof some of the type XVIII nucleosides have already discussed earlier.

[0550] The following working examples provide a further understanding ofthe method of the present invention. These examples are of illustrativepurposes, and are not meant to limit the scope of the invention.Equivalent, similar or suitable solvents, reagents or reactionconditions may be substituted for those particular solvents, reagents orreaction conditions described without departing from the general scopeof the method.

EXAMPLES

[0551] Melting points were determined in open capillary tubes on anElectrothermal digit melting point apparatus and are uncorrected. The UVabsorption spectra were recorded on an Uvikon 931 (KONTRON)spectrophotometer in ethanol. ¹H-NMR spectra were run at roomtemperature with a Varian Unity Plus 400 spectrometer. Chemical shiftsare given in ppm downfield from internal tetramethylsilane as reference.Deuterium exchange, decoupling experiments or 2D-COSY were performed inorder to confirm proton assignments. Signal multiplicities arerepresented by s (singlet), d (doublet), dd (doublet of doublets), t(triplet), q (quadruplet), br (broad), m (multiplet). All J-values arein Hz. FAB mass spectra were recorded in the positive-(FAB>0) ornegative-(FAB<0) ion mode on a JEOL DX 300 mass spectrometer The matrixwas 3-nitrobenzyl alcohol (NBA) or a mixture (50:50, v/v) of glyceroland thioglycerol (GT). Specific rotations were measured on aPerkin-Elmer 241 spectropolarimeter (path length 1 cm) and are given inunits of 10⁻¹deg cm² g⁻¹. Elemental analyses were performed by AtlanticMicrolab Inc. (Norcross, GA). Analyses indicated by the symbols of theelements or functions were within ±0.4% of theoretical values. Thinlayer chromatography was performed on Whatman PK5F silica gel plates,visualization of products being accomplished by UV absorbency followedby charring with 10% ethanolic sulfuric acid and heating. Columnchromatography was carried out on Silica Gel (Fisher, S733-1) atatmospheric pressure.

Example 1

[0552]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetylcytosine(2, R═H).

[0553] To a suspension of N⁴-acetylcytidine (5.7 g, 0.02 mol) inacetonitrile (300 mL) is added acetyl bromide (15 mL, 0.2 mol) over 30minutes under reflux. The mixture is refluxed for 4 hours, and thenconcentrated in vacuo to dryness. The residue is dissolved in methylenechloride (150 mL) and washed with water (150 mL). The organic layer isdried (Na₂SO₄), evaporated, and the residue crystallized from ethanol togiveI-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetylcytosine(2, R═H, 3.4 g, 40%), mp 179-180° C. ¹H NMR (CDCl₃) δ: 10.2 (bs, 1H,NHAC), 8.1 (d, 1H, H-6, 35,6=7.5 Hz), 7.5 (d, 1H, H-5, J_(5,6)=7.5 Hz),6.0 (d, 1H, H-1′, J_(1′,2′)<1 Hz), 5.5 (d, 1H, H-2′, J_(1′,2′)<1,J_(2′,3′)=0 Hz), 4.2-4.7 (m, 4H, H-3′,4′,5′,5″), 2.0-2.4 (3s, 9H, 3Ac).

[0554] In a similar manner but using the corresponding N-acylatedcytidine, the following nucleosides and their L-counterparts areprepared:

[0555]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-fluorocytosine,

[0556]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-chlororocytosine,

[0557]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-bromocytosine,

[0558]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-iodocytosine,

[0559]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-ethylcytosine,

[0560]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-n-propylcytosine,

[0561]1-(2,5-Di-O-acetyl-3-bromo-31-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-i-propylcytosine,

[0562]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-vinylcytosine,

[0563]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-(2-chlorovinyl)cytosine,

[0564]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-(2-bromovinyl)cytosine,

[0565]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-(2-iodovinyl)cytosine,

[0566]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N4-acetyl-5-(2-methoxylcarbonyl-vinyl)-cytosine,

[0567]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-(2-hydroxycarbonyl-vinyl)-cytosine,

[0568]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-phenylcytosine,

[0569]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetyl-5-benzylcytosine,

[0570]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xyloftiranosyl)-N⁴-benzoylcytosine,

[0571]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-fluorocytosine,

[0572]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofiiranosyl)-N⁴-benzoy1-5-cromocytosine,

[0573]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-chlrorocytosine,

[0574]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-bromocytosine,

[0575]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-methylcytosine,

[0576]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-ethylcytosine,

[0577]1-(2,5-Di-O-aceyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-ethylcytosine,

[0578]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-n-propylcytosine,

[0579]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5--i-poylcytosine,

[0580]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-(2-chlorovinyl)cytosine,

[0581]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-(2-bromovinyl)cytosine,

[0582]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-(2-iodovinyl)cytosine,

[0583]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-(2-methoxylcarbonyl-vinyl)cytosine,

[0584]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-(2-hydroxycarbonyl-vinyl)cytosine,

[0585]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-phenylcytosine,

[0586]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-benzoyl-5-benzylcytosine,

[0587]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoylcytosine,

[0588]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-chluorocytosine,

[0589]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-chlrorocytosine,

[0590]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-bromocytosine,

[0591]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-methycytosine,

[0592]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-methylcytosine,

[0593]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴anisoyl-5-nproylcytosine,

[0594]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴anisoyl-5-i-propylcytosine,

[0595]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-vipoylcytosine,

[0596]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-minylcytosine,

[0597]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-(2-chlorovinyl)cytosine,

[0598]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-(2-cbromovinyl)cytosine,

[0599]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-(2-iodovinyl)cytosine,

[0600]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-(2methoxylcarbonincoin yl)-cytosine,

[0601]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-(2-mehydroxycarbonyl-vinyl)-cytosine,

[0602]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-phenylcytosine,and

[0603]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-anisoyl-5-benzylcytosine.

Example 2

[0604]1-(2,5-Di-O-acetyl-3-deoxy-β-erythropentofiuranosyl)-N⁴-acetylcytosine.

[0605]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetylcytosine(2.15 g, 5 mmol) in 50% aqueous methanol (100 mL) is hydrogenated in aParr apparatus in the presence of powdered calcium carbonate (1 g) andPd-BaSO₄ catalyst (0.5 g) at the initial pressure of 45 psi. Thecatalyst is removed by filtration, and the filtrate is concentrated invacuo. The residue is crystallized from ethanol to give1-(2,5-di-O-acetyl-3-deoxy-P3-D-erythropentofuranosyl)-N⁴-acetylcytosine(3, R═H, 1.06 g, 60%), mp 174-177° C. ¹H NMR (CDCl₃) δ: 10.30 (bs, 1H,NHAc), 8.05 (d, 1H, H-6, J5,6=7.5 Hz), 7.43 (d, 1H, H-5, J5,6=7.5 Hz),5.90 (d, 1H, H-1′, J1′,2′=1.0 Hz), 5.46 (m, 1H, H-2′), 4.30-4.80 (3H,nm, H-4′,5′,5″), 2.10, 2.27 (2s, 9H, 3Ac), 1.60-2.00 (m, 2H, H-3′,3″).

[0606] In a similar manner but using the corresponding 3′-bromo-xylonucleosides, the following nucleosides and their L-counterparts areprepared:

[0607]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-benzoylcytosine,

[0608]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-benzoyl-5-methylcytosine,

[0609]1-(2,5-Di-O-acetyl-3-deoxy-βD-erythropentofuranosyl)-N⁴-benzoyl-5-ethylcytosine,

[0610]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythiropentofuranosyl)-N⁴-benzoyl-5-n-propylcytosine,

[0611]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-benzoyl-5-i-propylcytosine,

[0612]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-benzoyl-5-phenylcytosine,

[0613]1(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-benzoyl-5-benzylcytosine,

[0614]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-anisoylcytosine,

[0615]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-anisoyl-5-methylcytosine,

[0616]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-anisoyl-5-ethylcylosine,

[0617]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-anisoyl-5-n-propylcytosine,

[0618]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-anisoyl-5-i-propylcytosine,

[0619]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-anisoyl-5-phenylcytosineand

[0620] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N4_anisoyl-5-benzylcytosine.

Example 3

[0621] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)cytosine (3, R═H).

[0622]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N⁴-acetylcytosine(4.31 g, 0.01 mol) is treated with saturated methanolic ammonia (100 mL)at 0° C. for 30 minutes, and then concentrated in vacuo below 35° C. Theresidue is crystallized from methanol to give1-(3-bromo-3-deoxy-β-D-xylofuranosyl)cytosine (3, R═H). The UV and ¹HNMR (D₂O) are consistent with the xylo-structure.

[0623] In a similar manner but using the corresponding N-acylatedcytidines, the following nucleosides and their L-counterparts areprepared:

[0624] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-fluorocytosine,

[0625] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-chlororocytosine,

[0626] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-bromocytosine,

[0627] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-iodocytosine,

[0628] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-methylcytosine,

[0629] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-ethylcytosine,

[0630] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-n-propylcytosine,

[0631] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-i-propylcytosine,

[0632] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-vinylcytosine,

[0633] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-(2-chorovinyl)cytosine,

[0634] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-(2-bromovinyl)cytosine,

[0635] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-(2-bodovinyl)cytosine,

[0636]1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-(2-aininocarbonylvinyl)cytosine,

[0637]1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)cytosine,

[0638] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-phenylcytosine and

[0639] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-benzylcytosine.

Example 4

[0640] 3′-Deoxyctidiine (4, R═H).

[0641]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-acetylcytosine(3, R H, 700 mg, 2 mmol) is dissolved in methanolic ammonia (20 mL,saturated at 0° C.) and the solution is kept overnight at roomtemperature. The solvent is removed by evaporation in vacuo, and theresidue is dissolved in ethanol (20 mL), and then the pH of the solutionis adjusted to 3 with 2N sulfuric acid. The precipitates are collectedand crystallized from water-ethanol to give 3′-deoxycytidine (4) ashemisulfate (408 mg, 74%). Mp 202-203° C. (decomp). ¹H NMR (D₂O) δ: 8.23(d, 1H, H-6, J_(5,6)=8.0 Hz), 6.27 (d, 1H, H-5, J_(5,6)=80 Hz), 5.84 (d,1H, H-1′, J_(1′,2′)=1.0 Hz), 4.6 (m, 1H, H-2′), 3.9 (m, 3H, H-4′,5′,5″),1.95-2.15 (m, 2H, H-2′,2″).

[0642] In a similar manner but using the corresponding acylated3′-deoxynucloesides, the following nucleosides and their L-counterpartsare prepared: 3′-deoxy-5-methylcytidine, 3′-deoxy-5-ethylcytidine,3′-deoxy-5-n-propylcytidine, 3′-deoxy-5-i-propylcytidine,3′-deoxy-5-phenylcytidine, and 3′-deoxy-5-benzylcytidine.

Example 5

[0643] 2′,5′-Di-O-acetyl-3′-deoxyuridine.

[0644] 2′,5′-Di-O-acetyl-3-deoxy-N⁴-acetylcytidine (1.06 g, 3 mol) isdissolved in 70% acetic acid, and the solution is gently refluxedovernight. After concentration of the mixture in vacuo, the residue iscrystallized from ethanol to give 2′,5′-di-O-acetyl-3′-deoxyuridine (660mg, 96%). ¹H NMR spectrum shows that it contains two acetyl groups, twomethylene groups and two olefinic protons.

[0645] In a similar manner but using the corresponding 3′-deoxycytidines(4), the following 2′,5′-di-O-acetyl-3′-deoxyuridines and theirL-counterparts are prepared: 2′,5′-Di-O-acetyl-3-deoxy-5-methyluridine,2′,5′-di-O-acetyl-3-deoxy-5-ethyluridine,2′,5′-di-O-acetyl-3-deoxy-5-n-propyluridine,2′,5′-di-O-acetyl-3-deoxy-5-i-propyluridine,2′,5′-di-O-acetyl-3-deoxy-5-phenylunrdine and2′,5′-di-O-acetyl-3-deoxy-5-benzyluridine.

[0646] In a similar manner but using the corresponding 3′-deoxy cytosinenucleosides (2), the following uracil nucleosides and theirL-counterparts are prepared:

[0647] 2,5′-Di-O-acetyl-3-deoxy5-fluorouridine,

[0648] 2′, 5′-Di-O-acetyl-3-deoxy-5-chlorouridine,

[0649] 2′, 5′-Di-O-acetyl-3-deoxy-5-bromouridine,

[0650] 2′,5′-Di-O-acetyl-3-deoxy-5-iodouridine,

[0651] 2′, 5′-Di-O-acetyl-3-deoxy-5-methyluridine,

[0652] 2′, 5′-Di-O-acetyl-3-deoxy-5-ethyluridine,

[0653] 2′, 5′-Di-O-acetyl-3-deoxy-5-n-propyluridine,

[0654] 2′,5′-Di-O-acetyl-3-deoxy-5-i-propyluridine,

[0655] 2′,5′-Di-O-acetyl-3-deoxy-5-vinyluridine,

[0656] 2′, 5′-Di-O-acetyl-3-deoxy-5-(2-chlorovinyl)uridine,

[0657] 2′, 5′-Di-O-acetyl-3-deoxy-5-(2-bromovinyl)uridine,

[0658] 2′,5′-Di-O-acetyl-3-deoxy-5-(2-iodovinyl)uridine,

[0659] 2′, 5′-Di-O-acetyl-3-deoxy-5-(2-methoxylcarbonylvinyl)uridine,

[0660] 2′, 5′-Di-O-acetyl-3-deoxy-5-(2-hydroxycarbonylvinyl)uridine,

[0661] 2′,5′-Di-O-acetyl-3-deoxy-5-phenyluridine and

[0662] 2′,5′-Di-O-acetyl-3-deoxy-5-benzyluridine.

Example 6

[0663] 1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)uracil (5,R═H).

[0664]1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-N4-acetylcytosine(2, R═H, R′═CH₃) (4.31 g, 0.01 mol) is dissolved in 70% acetic acid, andthe solution is gently refluxed for 4 hours. After concentration of themixture in vacuo, the residue is crystallized from ethanol to give2′,5′-di-O-acetyl-3′-bromo-3′-deoxyuridine (5, 2.80 g, 91%). ¹H NMRspectrum shows that it contains two acetyl groups, two methylene groupsand two olefinic protons.

[0665] In a similar manner but using the corresponding2′,5′-di-O-acetyl-3′-bromo-3′-deoxy-N⁴-acylcytidines (2), the following1,5-di-O-acetyl-3′-bromo-3′-deoxyuridines and their L-counterparts areprepared:

[0666] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-fluorouracil,

[0667] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-chlororouracil,

[0668] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-bromouracil,

[0669] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-iodouracil,

[0670] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-methyluracil,

[0671] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-ethyluracil,

[0672] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-n-propyluracil,

[0673] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-i-propyluracil,

[0674] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-vinyluracil,

[0675]1-(2,5-Di-O-acetyl3-deoxy-β-D-xylofuranosyl)-5-(2-chlorovinyl)uracil,

[0676]1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-(2-bromovinyl)uracil,

[0677]1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-(2-iodovinyl)uracil,

[0678]1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)uracil,

[0679]1-(2,5-Di-O-acetyl-3-deoxy-βD-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)uracil,

[0680] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-phenyluracil and

[0681] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-xylofuranosyl)-5-benzyluracil.

Example 7

[0682] 3′-Deoxyuridine (6b, R═H).

[0683] 2′,5′-Di-O-acetyl-3′-deoxyuridine (1.06 g, 3 mol) is dissolved inmethanolic ammonia (10 mL, saturated at 0° C.) overnight. Afterconcentration of the mixture in vacuo, the residue is crystallized fromethanol to give 3′-deoxyuridine (6b, 660 mg, 96%).

[0684] In a similar manner but using the corresponding acylated3′-deoxy-uracil nucleosides (6b) or their L-counterparts, the followingnucleosides are prepared: 3-Deoxy-5-methyluridine,3-deoxy-5-ethyluridine, 3-deoxy-5-n-propyluridine,3-deoxy-5-i-propyluridine, 3-deoxy-5-phenyluridine, and3-deoxy-5-benzyluridine.

Example 8

[0685] 1-(3-Bromo-3-deoxy-fD-xylofuranosyl)uracil (6a, R═H).

[0686] 1-(2′,5′-Di-O-acetyl-3′-bromo-3′-deoxy-β-D-xylofuranosyl)uracil(5, R═H) is dissolved in methanolic ammonia (10 mL, saturated at 0° C.).After 1 hour at 0° C., the mixture is concentrated in vacuo, and theresidue is crystallized from ethanol to give 3′-bromo-3′-deoxyuridine(6a, 660 mg, 96%). The UV and ¹H NMR are consistent with the structure.

[0687] In a similar manner but using the corresponding acylated3′-bromo-xylosyluracils, the following nucleosides and theirL-counterparts are prepared:

[0688] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-fluorouracil,

[0689] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-chlororouracil,

[0690] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-bromouracil,

[0691] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-iodouracil,

[0692] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-methyluracil,

[0693] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-ethyluracil,

[0694] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-n-propyluracil,

[0695] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-i-propyluracil,

[0696] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-vinyluracil,

[0697] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-(2-chlorovinyl)uracil,

[0698] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-(2-bromovinyl)uracil,

[0699] 1-(3-Bromo-3deoxy-β-D-xylofuranosyl)-5-(2-iodovinyl)uracil,

[0700]1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-(2-aminocarbonylvinyl)uracil,

[0701]1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)uracil,

[0702] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-phenyluracil and

[0703] 1-(3-Bromo-3-deoxy-β-D-xylofuranosyl)-5-benzyluracil.

Example 9

[0704] 2′,5-Di-O-triphenylmethyluridine (7, R═H).

[0705] A mixture of uridine (24.4 g, 0.1 mol) and triphenylchloromethane(83.5 g, 0.3 mol) in anhydrous pyridine (250 mL) is stirred overnight atroom temperature, and then is refluxed for 4 hours. After cooling toroom temperature, the mixture is poured into water with vigorousstirring. The water is removed by decantation, and the gummy residue istreated with water, stirred and the water decanted. This process isrepeated several times, after which the residue is treated with hotwater (500 mL), stirred and the water decanted. This process is repeatedtwice. The residue is dissolved in methylene chloride, dried (Na₂SO₄),and concentrated in vacuo. The residue is dissolved in minimum amount ofbenzene, and the solution diluted with ethyl ether to turbidity, and themixture left overnight at 15° C. The precipitates are collected andrecrystallized from benzene-ethyl ether to give 7 (R H) (22.8 g, 31%),mp 224-225° C. The combined fitrates are concentrated, and the residuedissolved in methylene chloride and chromatographed over a silica gelcolumn using methylene chloride-ethanol (99:1 v/v), (98:2 v/v) and (97:3v/v). Compound 7 is eluted first (10 g, 14%), followed by3′,5′-di-O-triphenylmethyluridine (3 1.0 g, 42.5%).

[0706] In a similar manner but using the corresponding nucleosides, thefollowing 2′,5′-di-O-protected and 3′,5′-di-O-protected nucleosides andtheir L-counterparts are prepared:

[0707] 2′, 5′-Di-O-triphenylmethyl-5-fluorouridine,

[0708] 2′, 5′-Di-O-triphenylmethyl-5-chlorouridine,

[0709] 2′, 5′-Di-O-triphenylmethyl-5-bromouridine,

[0710] 2′,5′-Di-O-triphenylmethyl-5-iodouridine,

[0711] 2′, 5′-Di-O-triphenylmethyl-5-methyluridine,

[0712] 2′,5′-Di-O-triphenylmethyl-5-ethyluridine,

[0713] 2′,5′-Di-O-triphenylmethyl-5-n-propyluridine,

[0714] 2′, 5′-Di-O-triphenylmethyl-5-i-propyluridine,

[0715] 2′,5′-Di-O-triphenylmethyl-5-vinyluridine,

[0716] 2′,5′-Di-O-triphenylmethyl-5-ethynyluridine,

[0717] 2′,5′-Di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,

[0718] 2′,5′-Di-O-triphenylmethyl-5-(2-bromovinyl)uridine,

[0719] 2′,5′-Di-O-triphenylmethyl-5-(2-iodovinyl)uridine,

[0720] 2′,5′-Di-O-triphenylmethyl-5-(2-methoxylcarbonylvinyl)uridine,

[0721] 2′,5′-Di-O-triphenylmethyl-5-(2-hydroxycarbonylvinyl)uridine,

[0722] 2′,5′-Di-O-triphenylmethyl-5-phenyluridine,

[0723] 2′,5′-Di-O-triphenylmethyl-5-benzyluridine,

[0724] 3′,5′-Di-O-triphenylmethyl-5-fluorouridine,

[0725] 3′,5′-Di-O-triphenylmethyl-5-chlorouridine,

[0726] 3′, 5′-Di-O-triphenylmethyl-5-bromouridine,

[0727] 3′,5′-Di-O-triphenylmethyl-5-bodouridine,

[0728] 3′,5′-Di-O-triphenylmethyl-5-methyluridine,

[0729] 3′,5′-Di-O-triphenylmethyl-5-ethyluridine,

[0730] 3′,5′-Di-O-triphenylmethyl-5-n-propyluridine,

[0731] 3′,5′-Di-O-triphenylmethyl-5-i-propyluridine,

[0732] 3′,5′-Di-O-triphenylmethyl-5-vinyluridine,

[0733] 3′, 5′-Di-O-triphenylmethyl-5-ethynyluridine,

[0734] 3′,5′-Di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,

[0735] 3′,5′-Di-O-triphenylmethyl-5-(2-bromovinyl)uridine,

[0736] 3′,5′-Di-O-triphenylmethyl-5-(2-iodovinyl)uridine,

[0737] 3′,5′-Di-O-triphenylmethyl-5-(2-methoxylcarbonylvinyl)uridine,

[0738] 3′,5′-Di-O-triphenylmethyl-5-(2-hydroxycarbonylvinyl)uridine,

[0739] 3′,5′-Di-O-triphenylmethyl-5-phenyluridine and

[0740] 3′,5′-Di-O-triphenylmethyl-5-benzyluridine.

Example 10

[0741] 3′-O-Mesyl-2,5′-di-O-triphenylmethyluridine (8, R═H).

[0742] To a cooled solution of 2′,5′-di-O-triphenylmethyluridine (7,R═H, 7.28 g, Immol) in pyridine (100 mL) is added drop wise mesylchloride (1 mL), and the reaction is kept overnight at 4° C. Thereaction is quenched by addition of ethanol (5 mL). After 2 hours ofstirring at room temperature, the mixture is concentrated in vacuo. Theresidue is triturated with ethanol (250 mL), and the solid collected,and recrystallized from ethanol to give 8 (R═H) (7.45 g, 92%), mp225-226° C.

[0743] In a similar manner but using the corresponding nucleosides, thefollowing 2′,5′-di-O-triphenylmethylated and3′,5′-di-O-triphenylmethylated nucleosides and their L-counterparts areprepared:

[0744] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-fluorouridine,

[0745] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-chlorouridine,

[0746] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-bromouridine,

[0747] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-iodouridine,

[0748] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-methyluridine,

[0749] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-ethyluridine,

[0750] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-n-propylunrdine,

[0751] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-i-propyluridine,

[0752] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-vinyluridine,

[0753] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-ethynyluridine,

[0754] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,

[0755] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-(2-bromovinyl)uridine,

[0756] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-(2-iodovinyl)uridine,

[0757]3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-(2-methoxylcarbonylvinyl)uridine,

[0758]3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-(2-hydroxycarbonylvinyl)uridine,

[0759] 3′-O-Mesyl-2′,5′-di-O-triphenylmethyl-5-phenyluridine,

[0760] 3′-O-Mesyl-2′,S ′-di-O-triphenylmethyl-5-benzyluridine,

[0761] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-fluorouridine,

[0762] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-chlorouridine,

[0763] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-bromouridine,

[0764] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-iodouridine,

[0765] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-methyluridine,

[0766] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-ethyluridine,

[0767] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-n-propyluridine,

[0768] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-i-propyluidine,

[0769] 2′-O-Mesyl3′,5′-di-O-triphenylmethyl-5-vinyluridine,

[0770] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-ethynyluridine,

[0771] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,

[0772] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-(2-bromovinyl)ue, dine,

[0773] 2′-O-Mesyl-3′,5′-di-triphenylmethyl-5-(2-iodovinyl)uridine,

[0774]2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-(2-methoxylcarbonylvinyl)uridine,

[0775] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-(2-hydroxycaibonylvinyl)uridine,

[0776] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-phenyluridine and

[0777] 2′-O-Mesyl-3′,5′-di-O-triphenylmethyl-5-benzyluridine.

Example 11

[0778]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-_(D)-xylofuranosyl)uracil (9,R═H X′═OH).

[0779] A mixture of 3′-O-mesyl-2′,5′-di-O-triphenylmethyluridine (806mg, 1 mmol), sodium benzoate (2 g) in dimethylformamide (40 mL) isheated at 130-140° C. overnight. The mixture is cooled to roomtemperature, and poured onto IL of water with stirring. The precipitatesare collected by decantation and triturated with ethanol (100 mL) togive 3′-anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)uracil (9,R H, X′═OH), (500 mg, 75%), mp 237° C.

[0780] In a similar manner but using the corresponding 5-substituted3′-O-mesyl-2′,5′-di-O-triphenylmethyluridines (8), the following2,3′-anhydro-di-O-triphenylmethylated nucleosides and theirL-counterparts are prepared:2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-fluorouracil.

[0781]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-chlorouridine,

[0782]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-bromouridine,

[0783]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-iodouridine,

[0784]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-methyluridine,

[0785]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-ethyluridine,

[0786]2,3′-Anhydro-1-(2,5-di-triphenylmethyl-β-D-xylofuranosyl)-5-n-propyluridine,

[0787]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-i-propyluridine,

[0788]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-vinyluridine,

[0789]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-ethynyluridine,

[0790] 2,3′-Anhydro-1-s

[0791]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-(2-bromovinyl)uridine,

[0792]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-(2-iodovinyl)uridine,

[0793]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)-uridine,

[0794]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)-uridine,

[0795]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-phenyluridineand

[0796]2,3′-Anhydro-1-(2,5-di-O-triphenylmethyl-β-D-xylofuranosyl)-5-benzyluridine.

[0797] In a similar manner but using the corresponding 5-substituted2′-O-mesyl-3′,5′-di-O-triphenylmethyluridines, the following2,2′-anhydro-3′,5′-di-O-triphenylmethylated nucleosides and theirL-counterparts are prepared:

[0798]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-fluorouracil,

[0799] 2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-chlorouridine,

[0800] 2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-bromouridine,

[0801] 2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-iodouridine,

[0802]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-methyluridine,

[0803]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-ethyluridine,

[0804] 2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-n-propyluridine,

[0805]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-i-propyluridine,

[0806]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-vinyluridine,

[0807]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-ethynyluridine,

[0808] 2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-(2-chlorovinyl)uridine,

[0809] 2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-(2-bromovinyl)uridine,

[0810]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-(2-iodoviny)uridine,

[0811]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-(2-methoxylcarbonylvinyl)-uridine,

[0812] 2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-(2-hydroxycarbonylvinyl)-uridine,

[0813]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-phenyluridineand

[0814]2,2′-Anhydro-1-(3,5-di-O-triphenylmethyl-β-D-arabinofuranosyl)-5-benzyluridine.

Example 12

[0815] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyluridine (11, R═H, X═I,X′═OH).

[0816] A mixture of 3′-O-mesyl-2′,5′-di-O-triphenylmethyluridine (8,1.61 g, 2 mmol), sodium iodide (3 g, 20 mmol) in 1,2-dimethoxyethane (40mL) is heated at reflux overnight. The solvent is removed by evaporationin vacuo, the residue is dissolved in methylene chloride. The solutionis washed successively with 5% sodium thiosulfate and water, dried oversodium sulfate, and concentrated to dryness in vacuo. The residue ischromatographed over a silica gel column using methylene chloride-ethylether (3:1 v/v) as the eluent to give 703 mg (42%) of3′-deoxy-3′-iodo-2′,5′-di-O-triphenylmethyluridine (11, R═H, X═I,X′═OH).

[0817] In a similar manner but using the corresponding 5-substituted3′-O-mesyl-2′,5′-di-O-triphenylmethyluridines (8), the following 3′-iododerivatives are and their L-counterparts prepared:

[0818] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-fluorouridine,

[0819] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-chlorouridine,

[0820] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-bromouridine,

[0821] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-iodoun′dine,

[0822] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-methyluridine,

[0823] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-ethyluridine,

[0824] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-n-propyluridine,

[0825] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-i-propyluridine,

[0826] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-vinyluridine,

[0827] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-ethynyluridine,

[0828]3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,

[0829]3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-(2-bromovinyl)uridine,

[0830]3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-(2-iodovinyl)uridine,

[0831]3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl5-(2-methoxylcarbonylvinyl)uridine,

[0832]3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-(2-hydroxycarbonylvinyl)uridine,

[0833] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-phenyluridine and

[0834] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyl-5-benzyluridine.

[0835] In a similar manner but using the corresponding 5-substituted2′-O-mesyl-3′,5′-di O-triphenylmethyluridines, the following 2′-iododerivatives and their L-counterparts are prepared:

[0836] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-fluorouridine,

[0837] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-chyorourbdine,

[0838] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-bromouridine,

[0839] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-iodouridine,

[0840] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-methyluridine,

[0841] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-ethyluridine,

[0842] 2′-Deoxy-2′-iodo-3′,5′-di-triphenylmethyl-5-n-propyluridine,

[0843] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-i-propyluridine,

[0844] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-vinyluridine,

[0845] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-ethynyluridine,

[0846]2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,

[0847]2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-(2-bromovinyl)uridine,

[0848]2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-(2-iodovinyl)uridine,

[0849]2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-(2-methoxylcarbonylvinyl)uridine,

[0850]2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-(2-hydroxycarbonylvinyl)uridine,

[0851] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-phenyluridine and

[0852] 2′-Deoxy-2′-iodo-3′,5′-di-O-triphenylmethyl-5-benzyluridine.

Example 13

[0853] 3′-Iodo-3′-deoxyuridine.

[0854] 3′-Deoxy-3′-iodo-2′,5′-di-O-triphenylmethyluridine (840 mg, 1mmol) (11, R═H, X═I, X′═OH) is dissolved in a 10:1 mixture of methylenechloride and trifluoroacetic acid (20 mL), and the mixture is kept atroom temperature. The solvent is removed in vacuo, and the residue istriturated with ethyl ether (15 mL×2). The ether-insoluble residue iscrystallized from methanol ether to give 3′-iodo-3′-deoxyuridine (312mg, 88.1%).

[0855] In a similar manner but using the corresponding 5-substituted3′-deoxy-3′-iodo-2′,5′-di-O-triphenylmethyluridines, the following3′-iodouridine derivatives and their L-counterparts are prepared:3′-Deoxy-3′-iodo-5-iluorouridine, 3′-deoxy-3′-iodo-5-chlorouridine,3′-deoxy-3′-iodo-5-bromo-uridine, 3′-deoxy-3′-iodo-5-iodouridine,3′-deoxy-3′-iodo-S-methyl-urndine, 3′-deoxy-3′-iodo-5-ethyluridine,3′-deoxy-3′-iodo-5-n-propyluridine, 3′-deoxy-3′-iodo-5-i-propyl-uridine,3′-deoxy-3′-iodo-5-vinyluridine, 3′-deoxy-3′-iodo-5-ethynyluri dine,3′-deoxy-3′-iodo-5-(2-chloro-vinyl)-uridine,3′-deoxy-3′-iodo-5-(2-bromovinyl) uridine,3′-deoxy-3′-iodo-5-(2-iodovinyl)uridine,3′deoxy-3′-iodo-5-(2-methoxylcarbonyl-vinyl)uridine,3′-deoxy-3′-iodo-5-(2-hydroxy-carbonyl-vinyl)-uridine,3′-deoxy-3′-iodo-5-phenyluridine, and 3′-deoxy-3′-iodo-5-benzyl-uridine.

[0856] In a similar manner but using the corresponding 5-substituted2′-deoxy-2′-iodo-3′,5′-di-O-triphenylmethyluridines, the following2′-iodouridine derivatives and their L-counterparts are prepared:2′-deoxy-2′-iodo-5-fluorouridine, 2′-deoxy-2′-iodo-5-chlorouridine,2′-deoxy-2′-iodo-5-bromo-uridine, 2′-deoxy-2′-iodo-5-iodouridine,2′-deoxy-2′-iodo-5-methyl-uridine, 2′-deoxy-2′-iodo-5-ethyluridine,2′-deoxy-2′-iodo-5-n-propyluridine, 2′-deoxy-2′-iodo-5-i-propyl-uridine,2′-deoxy-2′-iodo-5-vinyluridine, 2′-deoxy-2′-iodo-5-ethynyluridine,2′deoxy-2′-iodo-5-(2-chlorovinyl)-uridine,2′-deoxy-2′-iodo-5-(2-bromovinyl)uridine,2′-deoxy-2′-iodo-5-(2-iodovinyl)uridine,2′-deoxy-2′-iodo-5-(2-methoxylcarbonylvinyl)uridine,2′-deoxy-2′-iodo-5-(2-hydroxycarbonyl-vinyl)-uridine,2′-deoxy-2′-iodo-5-phenyluridine, and 2′-deoxy-2′-iodo-5-benzyluridine.

Example 14

[0857] 9-(2-O-Acetyl-3-bromo-3-deoxy-,8-D-xylofuranosyl)adenitne (14,R═H, X′═Br, Y═NH₂, Z=H).

[0858] Compound 14 (R=2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl, X═Br,Y═NH₂, Z=H, 500 mg, I mmol) is dissolved in methanolic hydrogen chlorideprepared by addition of 3 drops of acetyl chloride in 10 mL of methanol.After 30 minutes at room temperature, 3 mL of saturated sodiumbicarbonate solution is added, and the mixture concentrated in vacuo todryness. The residue is triturated with ethanol until supernatant doesnot show significant UV absorption at 260 nm, The ethanol extracts areconcentrated, and the residue is crystallized from methanol to give thedesired 14 (R═H, X═Br, Y═NH₂, Z=H), 325 g (87%). ¹H NMR (D₆-DMSO) δ:8.16, 8.32 (2s, H-2 and H-8), 6. 10 (d, 1H, H-1′, J_(1′,2′)=3.9 Hz),5.91 (dd, 1H, H-2′, J_(1′,2′)=3.9, J_(2′,3′)=4.1 Hz), 5.85 (dd, 1H,H-3′, J_(2′,3′)=4.1, J_(3′,4′)=5.1 Hz), 4.38 (dt, 1H, H-4′,J_(3′,4′)=5.1, J_(4′,5′)=J_(4′,5)=5.0 Hz), 3.79 (dd, 2H, H-5′, 5″), 2.09(s, 3H, Ac).

[0859] In a similar manner but using the corresponding purinenucleosides, the following 2′-O-acetyl-3′-bromo-3′-deoxy-D-xylonucleosides (14) and their L-counterparts are prepared:

[0860] 9-(2-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)guanine,

[0861] 9-(2-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-6-chloropurine,

[0862]9-(2O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-2,6-dichloropurine,

[0863]9-(2-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-2-amino-6-chloropurine,

[0864]9-(2-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-6-methylthiopurine and

[0865] 9-(2-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)-6-methoxypurine.

Example 15

[0866] 9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-xylofuranosyl]adenine (14,R=2,5,5-trimethyl-1,3-dioxalan-2-one-2-yl, X═Br, Y═NH₂, Z═H).

[0867] A mixture of adenosine (13, Y═NH₂, Z═H, 10 g, 0.037 mol) andα-acetoxy-isobutyryl bromide (24 g, 0.117 mol) in acetonitrile (120 mL)is stirred at room temperature for 45 minutes. The solvent is removed invacuo, and the residue is dissolved in ethyl acetate, washed with sodiumbicarbonate solution and water, dried over sodium sulfate, andconcentrated in vacuo. The residue is crystallized from methanol to give6.5 g (35%) of 14 (X═Br, Y═NH₂, Z═H), mp 169-170° C. ¹H NMR (D₆-DMSO) δ:8.17, 8.26 (2s, 1H each, H-2 and H-6), 6.16 (d, 1H, H-1′, J_(1′,2′)=3.5Hz), 5.94 (dd, 1H, H-2′, J_(1′,2′)=3.5 Hz, J_(2′,3′)=3.0 Hz), 4.92 (dd,1H, H-3′, J_(2′,3′)=3.0 Hz, J_(3′,4′)=4.8 Hz), 4.54 (m, 1H, H-4′), 3.94(m, 2H, H-5′,5″), 2.10 (s, 3H, Ac), 1.73, 1.58, 1.47 (3s, 3H each, CH₃groups on 5′). The mother liquor of crystallization of 14 contains amixture of 2′-bromo-2′-deoxy-D-arabinosyl isomer 15, as judged by ¹HNMR.

[0868] In a similar manner but using the corresponding purinenucleosides, the following 3′-bromo-3′-deoxy derivatives (14) and theirL-counterparts are prepared:

[0869]9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-xylofuranosyl]-guanine,

[0870]9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-xylofuranosyl]-6-chloropurine,

[0871]9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-xylofuranosyl]-2,6-dichloropurine,

[0872]9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-xylofuranosyl]-2-amino-6-chloropurine,

[0873]9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-xylofuranosyl]-6-methylthiopurine,

[0874]9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-xylofuranosyl]-6-methoxypurine,

[0875] 9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-arabino-furanosyl]guanine,

[0876]9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-arabino-furanosyl]-6-chloropurine,

[0877]9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-arabino-furanosyl]-2,6-dichloropurine,

[0878]9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-arabino-furanosyl]-2-amino-6-chloropurine,

[0879]9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-arabino-furanosyl]-6-methylthiopurineand

[0880] (i)9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-arabino-furanosyl]-6-methoxypurine.

Example 16

[0881] 2′, 3′-Anhydroadenosine (18, Y═NH₂, Z═H).

[0882]9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl)-β-D-xylo-furanosyl]adenine14 (5.0 g, 0.01 mol) is treated with 1M sodium methoxide in methanol (20mL) for 1 hour at room temperature. The mixture is neutralized withglacial acetic acid, and is kept refrigerator overnight. Crystalline 18deposited is collected by filtration, 2.1 g (84%). ¹H NMR spectrum ofthis sample is identical with the one prepared by an alternativeprocedure by Mendez, E. et al. J. Virol. 1998, 72, 4737.

[0883] In a similar manner but using the corresponding purinenucleosides, the following 2′,3′-anhydro-β-ribo derivatives (18) andtheir L-counterparts are prepared: 2′,3′-anhydroguanosine,9-(2,3-anhydro-β-D-ribofuranosyl]-6-methylmercaptopurine, and9-(2,3-anhydro-β-D-ribo-furanosyl]-2-amino-6-methoxypurine.

Example 17

[0884] 9-(3-Deoxy-3-iodo-β-D-xylofuranosyl)adenine (19, X═I, Y═NH₂,Z═H).

[0885] A mixture of 18 (Y═NH₂, Z═H, 1 g, 4 mmol), sodium iodide (1.5 g,10 mmol), sodium acetate (100 mg) and acetic acid (5 mL) in butanone (30mL) is gently refluxed for 3 hours. Evaporation of the solvent in vacuo,and trituration of the residue with water afford 19 (X═I, Y═NH₂, Z═H),1.2 g (80%). ¹H NMR (D₆-DMSO) δ: 8.24, 8.34 (2s, 1H each, H-2 and H-8),5.90 (d, 1H, H-1′, J_(1′,2′)=4.7 Hz), 4.96 (dd, 1H, H-2′, J_(1′,2′)=4.7,J_(2′,3′)=4.9 Hz), 4.60 (dd, 1H, H-3′, J_(2′,3′)=4.9, J_(3′,4′)=4.7 Hz),4.80 (d, 2H, H-5′,5″), 4.40 (m, 1H, H-4′).

[0886] In a similar manner but using the corresponding2′,3′-anhydro-D-ribo purine nucleosides (14), the following3′-deoxy-3′-iodo-D-xylo nucleosides and their L-counterparts areprepared:

[0887] 9-(3-Deoxy-3-iodo-β-D-xylofuranosyl)guanine,

[0888] 9-(3-Deoxy-3-iodo-β-D-xylofuranosyl)-6-methylmercaptopurine,

[0889] 9-(3-Deoxy-3-iodo-β-D-xylofuranosyl)-6-methoxypurine,

[0890]9-(3-Deoxy-3-iodo-β-D-xylofuranosyl)-2-amino-6-methylmercaptopurine and

[0891] 9-(3-Deoxy-3-iodo-β-D-xylofuranosyl)-2-amino-6-methoxypurine.

Example 18

[0892] 3′-Deoxyadenosine (20, Y═NH₂, Z═H).

[0893] A solution of 19 (Y═NH₂, Z═H, 380 mg, 1 mmol) in methanol (75 mL)is shaken in an atmosphere of hydrogen in the presence of 5% Pd/BaSO₄catalyst (100 mg) and triethylamine (1 mL) at the initial pressure of 3atm overnight. After removal of the catalyst, the solvent is evaporatedin vacuo, and the residue is crystallized from methanol to give3′-deoxyadenosine 20 (Y═NTH₂, Z═H), 200 mg (80%). The ¹H NMR spectrum ofthis sample is identical with that of cordycepin.

[0894] In a similar manner but using the corresponding 3′-iodo-D-xylopurine nucleosides (19), the following 3′-deoxy-nucleosides and theirL-counterparts are prepared:9-(3-Deoxy-β-D-erythropentofuranosyl)guanine,9-(3-deoxy-β-D-erythropentofuranosyl) purine,9-(3-deoxy-β-D-erythropentofuranosyl)-6-methoxypurine,9-(3-deoxy-β-D-erythropento-furanosyl)-2-amino-purine and9-(3-deoxy-β-D-erythropentofuranosyl)-2-amino-6-methoxypurine.

Example 19

[0895] 3-(β-D-Ribofuranosyl)-8-azaxanthine (24, X═OH, Y═N).

[0896] To a solution of 5-nitrouridine (300 mg) in DMF (60 mL) is addedsodium azide (100 mg), and the mixture is stirred overnight at roomtemperature. The solvent is removed in vacuo, and the residue isdissolved in minimal amount of hot water and the pH adjusted to 3-4 withdiluted hydrochloric acid. The precipitates are recrystallized fromwater, mp 164-166° C. (dec). anal Calcd for C₉H₁₁N₅O₆H₂O : C, 35.64; H,4.29; N, 23.1. Found: C, 35.96; H, 4.01; N, 23.43.

Example 20

[0897]1,2-O-Isopropylidene-5-O-methoxycarbonyl-3-O-phenoxythiocarbonyl-α-D-xylofuranose(26, R═Ph).

[0898] To a solution of1,2-O-isopropylidene-5-O-methoxycarbonyl-cc-D-xylofuranose (25, 25.0g,0.1 mol) and 4-dimethylaminopyridine (25 g, 0.2 mol) in dry pyridine(250 mL) is added drop wise a solution of phenyl chlorothionoformate (50g, 0.3 mol) in acetonitrile (100 mL), and the reaction mixture isstirred at 50-60° C. for 24 hours. The solution is concentrated invacuo, and the residue is partitioned between methylene chloride andwater. The organic layer is washed successively with water, 0.1N sodiumhydroxide, water, 0.1N hydrochloric acid and water, and dried oversodium sulfate, and concentrated in vacuo to give 26 (R═Ph) as a syrupin quantitative yield (38.2 g). This syrup is used directly in the nextstep.

Example 21

[0899]3-Deoxy-1,2-O-isopropylidene-5-O-methoxycarbonyl-β-D-erythropentofuranose(27).

[0900] A solution of tri-n-butyltin hydride (58 g, 0.2 mol) in toluene(300 mL) is added over a period of 3 hours to a refluxing solution ofcompound 26 (R═Ph) above (19.2 g, 50 mmol) and2,2′-azobisisobutyronitrile (2.5 g, 15 mmol) in toluene (400 mL). Themixture is concentrated in vacuo, and the residue is dissolved inacetonitrile (300 mL), and the solution is extracted with petroleumether (4×100 mL) to remove tri-n-butyltin derivatives. The acetonitrilelayer is concentrated. The thin layer chromatography of the residueshows one major spot and ¹H NMR spectrum indicates the presence of threemethyl groups and no aromatic protons but contamination of a smallamount of butyltin derivatives. Without further purification, thisproduct is used in the next step.

Example 22

[0901]1,2-Di-O-acetyl-3-deoxy-5-O-methoxycarbonyl-D-erythropentofuranose (28).

[0902] To a stirred solution of 23 (2.32 g, 0.01 mol) in a mixture ofacetic acid (60 mL) and acetic anhydride (6 mL) is added drop wiseconcentrated sulfuric acid (3 mL) with ice-cooling at such a rate thatthe temperature is maintained at 15-25° C. After standing overnight atroom temperature, ice (250 g) is added to the solution, and then themixture is extracted with methylene chloride (3×50 mL). The combinedextracts are washed with saturated sodium bicarbonate solution (3×30mL), dried over sodium sulfate, and concentrated in vacuo to give 28(2.8 g, 100%) as an anomeric mixture. This compound is sufficiently pureto be used in the next step without further purification.

Example 23

[0903]1-(2-O-acetyl-3-deoxy-5-O-methoxycarbonyl-pBD-erythropentofuranosyl)-5-fluorouracil(29, X═OH, Z═F).

[0904] A mixture of 5-fluorouracil (2.6 g, 0.02 mol), ammonium sulfate(ca. 30 mg) in hexamethyldisilazane (15 mL) is refluxed until a clearsolution is obtained. The solvent is removed in vacuo, and the residueis dissolved in 1,2-dichloroethane (20 mL), and1,2-di-O-acetyl-3-deoxy-5-O-methoxycarbonyl-D-erythropentofuranose (28,5.5 g, 0.02 mol) in 1,2-dichloroethane (20 mL) is added. To the solutionis added tin tetrachloride (5.2 g, 0.02 mol), and the mixture is stirredovernight at room temperature, then is heated for 3 hours at 40-50° C.for 3 hours. Saturated sodium bicarbonate solution (40 mL) is added andstirred until carbon dioxide evolution ceases. The mixture is filteredthrough a Celite pad. The organic layer is separated, washed carefullywith saturated sodium bicarbonate solution (20 mL×2) and water (20mL×2), dried over sodium sulfate, and concentrated to dryness in vacuo.The residue is crystallized from ethanol to give 29 (4.3 g, 62%).

[0905] In a similar manner but using the corresponding pyrimidine bases,the following 2′,5′-protected 3′-deoxy-nucleosides and theirL-counterparts are prepared:

[0906]1-(2-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-chlorouracil,

[0907]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-,p-D-erythropentofuranosyl)-5-bromouracil,

[0908]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofaranosyl)-5-iodouracil,

[0909]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-cyanouracil,

[0910]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-ethoxycarbonyl-uracil,

[0911]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-aminocarbonyl-uracil,

[0912]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erylhropentofuranosyl)-5-acetyluracil,

[0913]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-methyluracil,

[0914]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-ethyluracil,

[0915]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-n-propyluracil,

[0916]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-i-propyluracil,

[0917]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-vinyluracil,

[0918]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-allyluracil,

[0919]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-ethynyluracil,

[0920]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-chlorovinyl)-uracil,

[0921]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-bromovinyl)-uracil,

[0922]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-iodovinyl)-uracil,

[0923]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-methoxylcarbonyl-vinyl)uracil,

[0924]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-hydroxycarbonyl-vinyl)uracil,1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-phenyluracil,

[0925]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-3-D-erythropentofuranosyl)-5-benzyluracil,1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-fluorocytosine,

[0926]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-chlorocytosine,

[0927]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-bromocytosine,

[0928]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-iodocytosine,

[0929]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-ethylcyanocytosine,

[0930]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5aiethoxycarbonyl-cytosine,

[0931]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-aminocarbonyl-cyeosine,

[0932]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-acetylcytosine,

[0933]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-methylcytosine,

[0934]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-ethylcytosine,

[0935]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-n-propylcytosine,

[0936]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-i-propyleytosine,

[0937]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-vinyicytosine,

[0938]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-allylcytosine,

[0939]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-ethynylcytosine,

[0940]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-chlorovinyl)-cytosine,

[0941]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-bromovinyl)-cytosine,

[0942]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-iodovinyl)-cytosine,

[0943]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-methoxyl-carbonylvinyl)cytosine,

[0944]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-(2-hydroxycarbonylvinyl)cytosine,

[0945]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-phenylcytosineand

[0946]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-5-benzylcytosine.

[0947] In a similar manner but using the corresponding pyrimidine andpurine bases, the following 2′,5′-di-O-acetyl 3′-deoxy-nucleosides andtheir L-counterparts are prepared:

[0948]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosy)-5-chlorouracil,

[0949]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-bromouracil,

[0950]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-iodouracil,

[0951]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-cyanouracil,

[0952]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-ethoxycarbonyluracil,

[0953]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-ethocarbonyluracil,

[0954]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-acetyluraci l ,

[0955]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-methyluracil,

[0956]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-ethyluracil,

[0957]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-n-propyluracil,

[0958]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-i-propyluracil,

[0959]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-vinyluracil,

[0960]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-allyluracil,

[0961]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-ethynyluracil,

[0962]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-chlorovinyl)uracil,

[0963]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-bromovinyl)uracil,

[0964]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-iodovinyl)uracil,

[0965]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofaranosyl)-5-(2-methoxylcarbonylvinyl)uracil,

[0966]1-(2,5-Di-O-acetyl-3-deoxy-β-D-crythropentofuranosyl)-5-(2-hydroxycarbonylvinyl)uracil,

[0967]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-phenyluracil,

[0968]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-benzyluracil,

[0969]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-fluorocytosine,

[0970]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-chlorocytosine,

[0971]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-bromocytosine,

[0972]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-iodocytosine,

[0973]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-cyanocytosine,

[0974]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-ethoxycarbonylcytosine,

[0975]1-(2,5-Di-O-acetyl-3-deoxy-βD-erythropentofuranosyl)-5-aminocarbonylcytosine,

[0976]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-acetylcytosine,

[0977]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erytfropentofiranosyl)-5-methylcytosine,

[0978] 1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-ethylcytosine,

[0979]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-n-propylcytosine,

[0980]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-i-propylcytosine,

[0981]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-vinylcytosine,

[0982]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-allylcytosine,

[0983]1-(2,5-Di-O-acetyL3-deoxy-β-D-erythropentofuranosyl)-5-ethynylcytosine,

[0984]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-cmhorovinyl)cytosine,

[0985]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-bromovinyl)cytosine,

[0986]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2idvinylcytosine,

[0987]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2methoxylcarbonylvinyl)cytosine,

[0988]1-(2,5Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-hydroxycarbonylvinyl)cytosine,

[0989] 1(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-phenylcytosine,

[0990]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-benzylcytosine,

[0991]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N6-benz:oyladenine,

[0992]1-(2,5-Di-O-acetyl-3deoxy-β-D-erythropentofuranosyl)-6-chloropurine,

[0993]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-2,6-dichloroputine,

[0994]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-6-methoxypurineand

[0995]1(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-6-methylmercaptopurine.

Example 24

[0996]1-(2-O-acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-6-chloropurine(30, X═Cl, Y═H).

[0997] A mixture of 6-chloropurine (3.1 g, 0.02 mol), ammonium sulfate(ca. 30 mg) in hexamethyldisilazane (25 mL) is refluxed until a clearsolution is obtained. The solvent is removed in vacuo, and the residueis dissolved in 1,2-dichloroethane (30 mL), and1,2-di-O-acetyl-3-deoxy-5-O-methoxycarbonyl-D-erythropentofuranose (28,5.5 g, 0.02 mol) in 1,2-dichloroethane (20 mL) is added. To the solutionis added tin tetrachloride (5.2 g, 0.02 mol), and the mixture is stirredovernight at room temperature, then is heated for 3 hours at 40-50° C.for 3 hours. Saturated sodium bicarbonate solution (50 mL) is added andstirred until carbon dioxide evolution ceases. The mixture is filteredthrough a Celite pad. The organic layer is separated, washed carefullywith saturated sodium bicarbonate solution (30 mL×2) and water (30mL×2), dried over sodium sulfate, and concentrated to dryness in vacuo.The residue is crystallized from ethanol to give 30 (4.3 g, 62%).

[0998] In a similar manner but using the corresponding purine bases, thefollowing 2′,5′-protected 3′-deoxy-nucleosides and their L-counterpartsare prepared:

[0999]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-N⁶benzoyladenine,

[1000]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-6-chloropurine,

[1001]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-2,6-dichloropurine,

[1002]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-2-acetamido-6-chloropurine,

[1003]1-(2-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-2-acetamido-6-methoxypurine,

[1004]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-6-methoxypurineand

[1005]1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-β-D-erythropentofuranosyl)-6-methylmercapto-purine.

Example 25

[1006] 1,2-O-Isopropylidene-5-O-t-butyldiphenylsilyl-α-D-xylofuranose(31).

[1007] A mixture of 1,2-O-isopropylidene-a-D-xylofuranose (38.0 g, 0.2mol), t-butyl-diphenylchlorosilane (70 g, 0.25 mol) and imidazole (21.5g, 0.4 mol) in N,N-dimethylformamide (50 mL) is stirred at roomtemperature for 1 hour. The solvent is removed in vacuo, and the residueis dissolved in ethyl acetate (1 L), and extracted with water (300 mL×2)and brine (300 mL), dried over sodium sulfate, and concentrated todryness in vacuo to give crude 31 (86 g, 100%), which is used directlyin the next step without further purification.

Example 26

[1008]1,2-O-Isopropylidene-3-O-mesyl-5-O-t-butyldiphenylsilyl-β-D-xylofuranose(32, R═Ms).

[1009] Mesyl chloride (17 g, 0.15 mol) is added drop wise to a solutionof crude 31 (43 g, 0.1 mol) in pyridine (100 mL), and the mixture iskept standing overnight at room temperature. Crashed ice (1 L) is addedto the mixture, and the product is extracted with methylene chloride(300 mL×3). The extracts are combined, washed with water (300 mL×2) andbrine (300 mL), dried over sodium sulfate, and concentrated in vacuo todryness. Traces of pyridine are removed by repeated azeotropicdistillation with toluene. The residue is dissolved in methylenechloride (500 mL) and washed with O.IN hydrochloric acid (250 mL×2) andwater, dried over sodium sulfate, and concentrated to dryness to givecrude 32 (R Ms), 50.1 g (99%). The ¹H NMR spectrum of this material issufficiently pure to be used directly in the next step.

Example 27

[1010] Methyl 3-O-mesyl-5-O-t-butyldiphenylsilyl-D-xylofuranoside (33,R═Ms).

[1011] A solution of crude 32 (50 g, 0.1 mol) in 1% anhydrous methanolichydrogen chloride (IL) is kept overnight at room temperature, and thenevaporated in vacuo to a syrup which is partitioned between water (100mL) and methylene chloride (150 mL). The organic layer is separated,washed with water (100 mL), dried over sodium sulfate, and concentratedin vacuo, giving crude 33, a syrup, weighing 48 g (100%). This materialis not further purified but used directly in the next step.

Example 28

[1012] Methyl 2,3-anhydro-5-O-t-butyldiphenylsilyl-D-fibofuranoside(34).

[1013] Crude 33 (48 g, 0.1 mol) is dissolved in methylene chloride (100mL) and treated with 2M methanolic sodium methoxide (60 mL), andrefluxed for 2 hours. Insoluble salt is removed by filtration, and thefiltrate is concentrated in vacuo to dryness. The residue is dissolvedin methylene chloride (150 mL), washed with water (100 mL×2), dried oversodium sulfate, and concentrated to dryness to give crude 30 (38 g,100%), which can be used directly in the next step without purification.

Example 29

[1014] Methyl 3-deoxy-3-iodo-5-O-t-butyldiphenylsilyl-D-ribofuranoside(35, X═I).

[1015] A mixture of 34 (38 g, 0.1 mol), sodium iodide (60 g, 0.4 mol),sodium acetate (0.6 g) and acetic acid (70 mL) in acetone (500 mL) isheated under reflux for 8 hours. The acetone is removed in vacuo, andthe residue is partitioned between methylene chloride (500 mL) and water(250 mL). The organic layer is separated, washed with 250 mL each ofwater, 0.1 M sodium thiosulfate solution, water and dried over sodiumsulfate. After removal of the solvent in vacuo, the residue iscrystallized from ethanol to afford 31 g (60.5%) of 35 (X═I).

Example 30

[1016] Methyl 3-deoxy-5-O-t-butyldiphenylsilyl-D-erythropentofuranoside(37, from 35).

[1017] Compound 35 (X═I, 25.6 g, 0.05 mol) is hydrogenated in ethylacetate (250 mL) with 5% palladium on charcoal (2 g). After theconsumption of hydrogen ceased, the mixture is filtered, and thefiltrate is washed with water (150 mL×2), dried over sodium sulfate, andconcentrated to dryness to give crude 37 (19 g, quantitative yield)which is sufficiently pure to be used directly in the next step.

Example 31

[1018] Methyl 3-deoxy-5-O-t-butyldiphenylsilyl-D-erythropentofuranoside(36, from 34).

[1019] A suspension of lithium aluminum hydride (8.4 g, 0.2 mol) in dryethyl ether (220 mL) is stirred under nitrogen atmosphere and cooled inan ice bath. To this suspension is added drop wise a solution of 34 (19g, 0.05 mol) in dry tetrahydrofuran (250 mL) at such a rate that thetemperature remains below 25° C. After 2 hours, another l g of lithiumaluminum hydride is charged, and the mixture is stirred overnight atroom temperature. The stirred mixture is cooled in an ice bath, andisopropanol (100 mL) is added drop wise, followed by acetone (50 mL).The mixture is concentrated in vacuo, and the residue is partitionedbetween ethyl ether (250 mL) and water (150 mL). Insoluble materials arefiltered through Celite pad which is washed with ether. The ether layeris separated, washed successively with 0.2N hydrochloric acid (150 mL×2)and water (150 mL×2), dried over sodium sulfate, and then concentratedto dryness to give crude 36 (16.5 g, 87%).

Example 32

[1020] Methyl 3-deoxy-D-erythropentofuranoside (38).

[1021] To a solution of crude 36 (13 g, 0.03 mol) in tetrahydrofuran(320 mL) is added drop wise IM solution of triethylammonium hydrogenfluoride (100 mL), and the mixture is stirred for 24 hours. The mixtureis concentrated in vacuo, and the residue is dissolved in water (200mL). Powdered calcium carbonate (20 g) is added, and the mixture isstirred overnight at room temperature, and then filtered. The filtrateis concentrated in vacuo to a syrup which is dissolved in chloroform(200 mL), filtered, and evaporated in vacuo to afford crude 38 (4.5 g,100%).

Example 33

[1022] 1,2,5-Tri-O-acetyl-3-deoxy-D-erythropentofuranose (38).

[1023] To a vigorously stirred mixture of crude methyl3-deoxy-D-erythropentofuranoside 37 (4.5 g, 0.03 mol) and acetic acid(80 mL) is added acetic anhydride (40 mL), followed by sulfuric acid (4mL), and the reaction mixture is stirred overnight at room temperature.The mixture is partitioned between methylene chloride (150 mL) andice-water (400 mL). The water layer is extracted with methylene chloride(100 mL×2). The combined organic layers are washed twice with equalvolumes of a saturated solution of sodium bicarbonate, once with water,dried over sodium sulfate, and concentrated to dryness in vacuo. Tracesof acetic acid are removed by several azeotropic distillations withtoluene to give crude 38 (5.1 g, 66%). The ¹H NMR spectrum shows thatthe major constituent of this product contains 3 acetyl groups and isthe β-anomer.

Example 34

[1024] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-fluorouraceil(3′-deoxy-5-fluorouridine,6b, X═OH, R═F).

[1025] A mixture of an acetyl derivative of 39 (X═OH, Z═F, 3.3 g, 0.01mol) and triethylamine (3 mL) in methanol (100 mL) is stirred overnightat room temperature. The mixture is concentrated in vacuo to dryness,and the residue is crystallized from ethanol to give3′-deoxy-5-fluorouridine (2.0 g, 83%), mp 169-171° C. ¹H NMR (D₆-DMSO)δ: 11.7 (bs, 1H, N³-H, exchangeable), 8.44 (d, 1H, H-6, J_(6,F)=7.1Hz),5.7 (d, 1H, 2′-OH, exchangeable), 5.5 (narrow m, 1H, H-i′), 5.3 (t,1H, 5′-OH, exchangeable), 4.1-4.5 (m, 2H, H-2′ and H-4′), 3.5-3.9 (m,2H, H-5′,5″), 1.6-2.2 (m, 2H, H-3′,3″).

[1026] In a similar manner but using the corresponding 2′,5′-di-O-acetylpyrimidine and purine nucleoside, the following 3′-deoxy-nucleosides andtheir L-counterparts are prepared:

[1027] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-chlorouracil,

[1028] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-bromouracil,

[1029] 1-(3-Deoxy—D-erythropentofuranosyl)-5-iodouracil,

[1030] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-cyanouracil,

[1031] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-ethoxycarbonyluracil,

[1032] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-aminocarbonyluracil,

[1033] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-acetyluracil,

[1034] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-methyluracil,

[1035] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-ethyluracil,

[1036] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-n-propyluracil,

[1037] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-i-propyluracil,

[1038] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-vinyluracil,

[1039] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-anlyluracil,

[1040] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-ethynyluracil,

[1041] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-(2-chlorovinyl)uracil,

[1042] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-(2-bromovinyl)uracil,

[1043] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-(2-iodovinyl)uracil,

[1044]1-(3-Deoxy-β-D-erythropentofuranosyl)-5-(2-methoxylcarbonylvinyl)uracil,

[1045]1-(3-Deoxy-β-D-erythropentofuranosyl)-5-(2-hydroxycarbonylvinyl)uracil,

[1046] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-phenyluracil,

[1047] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-benzyluracil,

[1048] 1-(3-Deoxy-β-D-erythropentofuranosyl)cytosine,

[1049] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-fluorocytosine,

[1050] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-chlorocytosine,

[1051] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-bromocytosine,

[1052] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-iodocytosine,

[1053] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-cyanocytosine,

[1054] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-ethoxycarbonylcytosine,

[1055] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-aminocarbonylcytosine,

[1056] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-acetylcytosine,

[1057] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-methylcytosine,

[1058] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-ethylcytosine,

[1059] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-n-propylcytosine,

[1060] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-i-propylcytosine,

[1061] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-vinylcytosine,

[1062] 1-(3-Deoxy-β-erythropentofuranosyl)-5-allycytosine,

[1063] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-ethynylcytosine,

[1064] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-(2-chlorovinyl)cytosine,

[1065] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-(2-bromovinyl)cytosine,

[1066] 1-(3Deoxy—D-erythropentofuranosyl)-5-(2-iodovinyl)cytosine,

[1067]1-(3-Deoxy-β-D-erythropentofuranosyl)-5-(2-methoxylcarbonylvinyl)cytosine,

[1068] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-(2-hydroxycarbonylvinyl)cytosine,

[1069] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-phenylcytosine,

[1070] 1-(3-Deoxy-β-D-erythropentofuranosyl)-5-benzylcytosine,

[1071] 1-(3-Deoxy-β-D-erythropentofuranosyl)-2-chloroadenine,

[1072] 1-(3-Deoxy-β-D-erythropentofuranosyl)-6-chloropurine,

[1073] 1-(3-Deoxy-β-D-erythropentofuranosyl)-2,6-dichloropurine,

[1074] 1-(3-Deoxy-β-D-erythropentofuranosyl)-2-acetamido-6-chloropurine,

[1075]1-(3-Deoxy-β-D-erythropentofuranosyl)-2-acetamido-6-methoxypurine,

[1076] 1-(3-Deoxy-β-D-erythropentofuranosyl)-6-methoxypurine and

[1077] 1-(3-Deoxy-β-D-erythropentofuranosyl)-6-methylmercaptopurine.

Example 35

[1078] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-fluorouracil.

[1079] A mixture of 5-fluorouracil (0.02 mol), ammonium sulfate (ca. 30mg) in hexamethyldisilazane (15 mL) is refluxed until a clear solutionis obtained. The solvent is removed in vacuo, and the residue isdissolved in 1,2-dichloroethane (20 mL), and1,2,5-tri-O-acetyl-3-O-mesyl-D-xylofuranose (5.5 g, 0.02 mol) in1,2-dichloroethane (20 mL) is added. To the solution is added tintetrachloride (5.2 g, 0.02 mol), and the mixture is stirred overnight atroom temperature, then is heated for 3 hours at 40-50° C. for 3 hours.Saturated sodium bicarbonate solution (40 mL) is added and stirred untilcarbon dioxide evolution ceases. The mixture is filtered through aCelite pad. The organic layer is separated, washed carefully withsaturated sodium bicarbonate solution (20 mL×2) and water (20 mL×2),dried over sodium sulfate, and concentrated to dryness in vacuo. Theresidue is crystallized from ethanol to give the title product (62%).The ¹H NMR spectrum of this sample is compatible with the structureindicated.

[1080] In a similar manner but using the corresponding pyrimidine andpurine bases, the following 2′,5′-di-O-acetyl 3′-substitutedxylo-nucleosides and their L-counterparts are prepared:

[1081] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-chlorouracil,

[1082] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-bromouracil,

[1083] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-iodouracil,

[1084] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-cyanouracil,

[1085]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-ethoxycarbonyluracil,

[1086]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-aminocarbonyluracil,

[1087] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-acetyluracil,

[1088] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-methyluracil,

[1089] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-ethyluracil,

[1090] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-n-propyluracil,

[1091] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-i-propyluracil,

[1092] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-vinyluracil,

[1093] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-allyluraeil,

[1094] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-ethynyluracil,

[1095]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-(2-chlorovinyl)uracil,

[1096]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-(2-bromovinyl)uracil,

[1097]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-(2-iodovinyl)uracil,

[1098]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)uracil,

[1099]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)uracll,

[1100] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-phenyluracil,

[1101] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-benzyluracil,

[1102] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosy)-5-fluorocytosine,

[1103] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-chlorocytosine,

[1104] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-bromocytosine,

[1105] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-iodocytosine,

[1106] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-cyanocytosine,

[1107]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-ethoxycarbonylcytosine,

[1108]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-ethnoxcarbonylcytosine,

[1109] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-ametylcytosine,

[1110] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-methylcytosine,

[1111] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-npoyleytosine,

[1112]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-i-propylcytosine,

[1113] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-ethylcytosine,

[1114]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-n-propylcytosine,

[1115]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-ethynylcytosine,

[1116] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-vinylcytosine,

[1117]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-(2-chlorovinyl)cytosine,

[1118]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-(2-bromovinyl)cytosine,

[1119]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)cytosine,

[1120]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)cytosine,

[1121] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-phenylcytosine,

[1122] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-benzylcytosine,

[1123]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-N⁶-benzoyladenine,

[1124] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-6-chloropurine,

[1125]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-2,6-dichloropurine,

[1126]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-2-acetamido-6-chloropurine,

[1127]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-2-acetamido-6-methoxypurine,

[1128] 1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-6-methoxypurine,

[1129]1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-6-methylmercaptopurine,

[1130] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-chlorouracil,

[1131] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-bromouracil,

[1132] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-iodouracil,

[1133] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-cyanouracil,

[1134]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-ethoxycarbonyluracil,

[1135]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-aminocarbonyluracil,

[1136] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-acetyluracil,

[1137] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-methyluracil,

[1138] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-ethyluracil,

[1139] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-n-propyluracil,

[1140] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-i-propyluracil,

[1141] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-vinyluracil,

[1142] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-allyluracil,

[1143] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-ethynyluracil,

[1144]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-chlorovinyl)uracil,

[1145]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-bromovinyl)uracil,

[1146]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-iodovinyl)uracil,

[1147]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)uracil,

[1148]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)uracil,

[1149] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-phenyluracil,

[1150] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-benzyluracil,

[1151] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-fluorocytosine,

[1152] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-chlorocytosine,

[1153] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-bromocytosine,

[1154] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-iodocytosine,

[1155] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-cyanocytosine,

[1156]1-(2,5-Di-O-acetyl-3-tosyl-β-D-xylofuranosyl)-5-ethoxycarbonylcytosine,

[1157]1-(2,5-Di-O-acetyl-3-tosyl-β-D-xylofuranosyl)-5-aminocarbonylcytosine,

[1158] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-acetylcytosine,

[1159] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-methylcytosine,

[1160] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-ethylcytosine,

[1161]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-n-propylcytosine,

[1162]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-i-propylcytosine,

[1163] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-vinylcytosine,

[1164] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-allylcytosine,

[1165]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-ethynylcytosine,

[1166]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-chlorovinyl)cytosine,

[1167]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-bromovinyl)cytosine,

[1168]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-iodovinyl)cytosine,

[1169]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)cytosine,

[1170]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)cytosine,

[1171] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-phenylcytosine,

[1172] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-5-benzylcytosine,

[1173] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-N-benzoyladenine,

[1174] 1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-6-chloropurine,

[1175]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-2,6-dichloropurine,

[1176]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosyl)-2-acetamido-6-chloropurine,

[1177]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylopentofuranosyl)-2-acetamido-6-methoxypurine,

[1178]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylopentofuranosyl)-6-methoxypurine and

[1179]1-(2,5-Di-O-acetyl-3-O-tosyl-β-D-xylofuranosy¹)-6-methylmercaptopurine.

Example 36

[1180] 1-(2,3, 5-Tri-O-acetyl-β-D-xylofuranosyl)thymine.

[1181] A mixture of thymine (0.02 mol), ammonium sulfate (ca. 30 mg) inhexamethyldisilazane (15 mL) is refluxed until a clear solution isobtained. The solvent is removed in vacuo, and the residue is dissolvedin 1,2-dichloroethane (20 mL), and 1,2,3,5-tri-O-acetyl-D-xylofuranose(5.5 g, 0.02 mol) in 1,2-dichloroethane (20 mL) is added. To thesolution is added tin tetrachloride (5.2 g, 0.02 mol), and the mixtureis stirred overnight at room temperature, then is heated for 3 hours at40-50° C. for 3 hours. Saturated sodium bicarbonate solution (40 mL) isadded and stirred until carbon dioxide evolution ceases. The mixture isfiltered through a Celite pad. The organic layer is separated, washedcarefully with saturated sodium bicarbonate solution (20 mL×2) and water(20 mL×2), dried over sodium sulfate, and concentrated to dryness invacuo. The residue is crystallized from ethanol to give product (4.3 g,62%). The ¹H NMR spectrum of this sample is compatible with thestructure indicated.

[1182] In a similar manner but using the corresponding pyrimidine andpurine bases, the following 2′,5′-di-O-acetyl 3′-substitutedxylo-nucleosides and their L-counterparts are prepared:

[1183] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-fluorouracil,

[1184] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-chlorouracil,

[1185] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-bromouracil,

[1186] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-iodouracil,

[1187] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-cyanouracil,

[1188] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-ethoxycarbonyluracil,

[1189] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-aminocarbonyluracil,

[1190] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-acetyluracil,

[1191] 2-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-methyluracil,

[1192] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-ethyluracil,

[1193] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-n-propyluracil,

[1194] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-i-propyluracil,

[1195] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-vinyluracil,

[1196] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-allyluracil,

[1197] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-ethynyluracil,

[1198] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-(2-chlorovinyl)uracil,

[1199] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-(2-bromovinyl)uracil,

[1200] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-(2-iodovinyl)uracil,

[1201] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)uracil,

[1202]1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)uracil,

[1203] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-phenyluracil,

[1204] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-benzyluracil,

[1205] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-fluorocytosine,

[1206] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-chlorocytosine,

[1207] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-bromocytosine,

[1208] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-iodocytosine,

[1209] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-cyanocytosine,

[1210]1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-ethoxycarbonylcytosine,

[1211] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-aminocarbonylcytosine,

[1212] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-acetylcytosine,

[1213] 1β-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-methycytosine,

[1214] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-ethylcytosine,

[1215] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-n-propylcytosine,

[1216] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-i-propylcytosine,

[1217] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-vinylcytosine,

[1218] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-allylcytosine,

[1219] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-ethynylcytosine,

[1220]1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-(2-chlorovinyl)cytosine,

[1221]1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-(2-bromovinyl)cytosine,

[1222] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)cytosine,

[1223] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-5-phenylcytosine,

[1224] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-N⁶-benzoyladenine,

[1225] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylofuranosyl)-6-chloropurine,

[1226] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-2,6-dichloropurine,

[1227]1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-2-acetamido-6-chloropurine,

[1228] 1-(2,3,5-Tri-O-acetyl-β-D-xylopentofuranosyl)-2-acetamido-6-methoxypurine,

[1229] 1-(2,3 ,5-Tri-O-acetyl-β-D-xylopentofuranosyl)-6-methoxypurineand

[1230] 1-(2,3,5-Tri-O-acetyl-β-D-xylofuranosyl)-6-methylmercaptopurine.

Example 37

[1231] 1-(3-Deoxy-3-O-mesyl-β-D-xylofuranosyl)-5-fluorouracil.

[1232] A mixture of1-(2,5-Di-O-acetyl-3-O-mesyl-β-D-xylofuranosyl)-5-fluorouracil (4.24 g,0.01 mol) in methanolic ammonia (100 mL) is stirred for 30 minutes at 0°C., and is concentrated in vacuo to dryness, and the residue iscrystallized from ethanol to give1-(3-deoxy-3-O-mesyl-β-D-xylofuranosyl)-5-fluorouracil (2.82 g, 83%). ¹HNMR (D₆-DMSO) showed that there is no acetyl group but one mesyl groupin the molecule.

[1233] In a similar manner but using the corresponding 2′,5′-di-O-acetylpyrimidine and purine nucleosides, the following 3′-O-mesyl-nucleosidesand their L-counterparts are prepared:

[1234] 1-(3-5-Mesyl-β-D-xylofuranosyl)-5-chlorouracil,

[1235] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-bromouracil,

[1236] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-iodouracil,

[1237] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-cyanouracil,

[1238] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-ethoxycarbonyluracil,

[1239] 2-(3-O-Mesyl-β-D-xylofuranosyl)-5-aminocarbonyluracil,

[1240] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-acetyluracil,

[1241] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-methyluracil,

[1242] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-ethyluracil,

[1243] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-n-propyluracil,

[1244] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-i-propyluracil,

[1245] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-vinyluracil,

[1246] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-allyluracil,

[1247] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-ethynyluracil,

[1248] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-(2-chlorovinyl)uracil,

[1249] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-(2-bromovinyl)uracil,

[1250] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-(2-iodovinyl)uracil,

[1251]1-(3-O-Mesyl-β-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)uracil,

[1252] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)uracil,

[1253] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-phenyluracil,

[1254] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-benzyluracil,

[1255] 1-(3-O-Mesyl-β-D-xylofuranosyl)cytosine,

[1256] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-fluorocytosine,

[1257] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-chlorocytosine,

[1258] 1-(3-O-Mesyl-β-D-xylofaranosyl)-5-bromocytosine,

[1259] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-iodocytosine,

[1260] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-cyanocytosine,

[1261] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-ethoxycarbonylcytosine,

[1262] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-aminocarbonylcytosine,

[1263] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-acetylcytosine,

[1264] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-methylcytosine,

[1265] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-ethylcytosine,

[1266] 1q-(3-Mesyl-β-D-xylofuranosyl)-5-n-propylcytosine,

[1267] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-i-propylcytosine,

[1268] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-vinylcytosine,

[1269] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-allylcytosine,

[1270] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-ethynylcytosine,

[1271] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-(2-chlorovinyl)cyto sine,

[1272] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-(2-bromovinyl)cytosine,

[1273] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-(2-iodovinyl)cytosine,

[1274]1-(3-O-Mesyl-3-D-xylofuranosyl)-5-(2-methoxylearbonylviny)cytosine,

[1275]1-(3-O-Mesyl-β-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)cytosine,

[1276] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-phenylcytosine,

[1277] 1-(3-O-Mesyl-β-D-xylofuranosyl)-5-benzylcytosine,

[1278] 1-(3-O-Mesyl-β-D-xylofuranosyl)-2-chloroadenine,

[1279] 1-(3-O-Mesyl-β-D-xylofuranosyl)-6-chloropurine,

[1280] 1-(3-O-Mesyl-β-D-xylofuranosyl)-2,6-dichloropurine,

[1281] 1-(3-O-Mesyl-β-D-xylofuranosyl)-2-acetamido-6-chloropurine,

[1282] 1-(3-O-Mesyl-β-D-xylofuranosyl)-2-acetamido-6-methoxypurine,

[1283] 1-(3-O-Mesyl-β-D-xylofuranosyl)-6-methoxypurine and

[1284] 1-(3-O-Mesyl-o-D-xylofuranosyl)-6-methylmercaptopurine.

Example 38

[1285] 1-β-D-Xylofuranosyl)-5-fluorouracil.

[1286] A mixture of1-(2,3,5-tri-O-acetyl-β-D-xylofuranosyl)-5-fluorouracil (3.88 g, 0.01mol) and triethylamine (3 mL) in methanol (100 mL) is stirred overnightat room temperature. The mixture is concentrated in vacuo to dryness,and the residue is crystallized from ethanol to giveI-(β-D-xylo-furanosyl)-5-fluorouracil (2.0 g, 76%). The UV and ¹H NMR(Me₂SO-d6) spectra of this sample are consistent with the productstructure.

[1287] In a similar manner but using the corresponding 2′,5′-di-O-acetylpyrimidine and purine bases, the following xylo-nucleosides and theirL-counterparts are prepared:

[1288] 1-(β-D-Xylofuranosyl)-5-chlorouracil,

[1289] 1-(β-D-Xylofuranosyl)-5-bromouracil,

[1290] 1-(3-D-Xylofuranosyl)-5-iodouracil,

[1291] 1-(β-D-Xylofuranosyl)-5-cyanouracil,

[1292] 1-(β-D-Xylofuranosyl)-5-ethoxycarbonyluracil,

[1293] 1-(β-D-Xylofuranosyl)-5-aminocarbonyluracil,

[1294] 1-(β-D-Xylofuranosyl)-5-acetyluracil,

[1295] 1-(β-D-Xylofuranosyl)-5-methyluracil,

[1296] 1-(β-D-Xylofuranosyl)-5-ethyluracil,

[1297] 1-(β-D-Xylofuranosyl)-5-n-propyluracil,

[1298] 1-(β-D-Xylofuranosyl)-5-i-propyluracil,

[1299] 1-(β-D-Xylofuranosyl)-5-vinyluracil,

[1300] 1-(β-D-Xylofuranosyl)-5-allyluracil,

[1301] 1-(β-D-Xylofuranosyl)-5-ethynyluracil,

[1302] 1-(β-D-Xylofuranosyl)-5-(2-chlorovinyl)uracil,

[1303] 1-(β-D-Xylofuranosyl)-5-(2-bromovinyl)uracil,

[1304] 1-(β-D-Xylofuranosyl)-5-(2-iodovinyl)uracil,

[1305] 1-(β-D-Xylofuranosyl)-5-(2-methoxylcarbonylvinyl)uracil,

[1306] 1-(β-D-Xylofuranosyl)-5-(2-hydroxycarbonylvinyl)uracil,

[1307] 1-(β-D-Xylofuranosyl)-5-phenyluracil,

[1308] 1-(β-D-Xylofuranosyl)-5-benzyluracil,

[1309] 1-(β-D-Xylofuranosyl)cytosine,

[1310] 1-(β-D-Xylofuranosyl)-5-fluorocytosine,

[1311] 1-(β-D-Xylofuranosyl)-5-chlorocytosine,

[1312] 1-(β-D-Xylofuranosyl)-5-bromocytosine,

[1313] 1-(β-D-Xylofuranosyl)-5-iodocytosine,

[1314] 1-(β-D-Xylofuranosyl)-5-cyanocytosine,1-(β-D-Xylofuranosyl)-5-ethoxycarbonylcytosine,

[1315] 1-(β-D-Xylofuranosyl)-5-aminocarbonylcytosine,

[1316] 1-(β-D-Xylofuranosyl)-5-acetylcytosine,

[1317] 1-(β-D-Xylofuranosyl)-5-methylcytosine,

[1318] 1-(β-D-Xylofuranosyl)-5-ethylcytosine,

[1319] 1-(β-D-Xylofuranosyl)-5-n-propylcytosine,

[1320] 1-(β-D-Xylofuranosyl)-5-i-propylcytosine,

[1321] 1-(β-D-Xylofuranosyl)-5-viny lcytosine,

[1322] 1-(β-D-Xylofuranosyl)-5-allylcytosine,

[1323] 1-(β-D-Xylofuranosyl)-5-ethynylcytosine,

[1324] 1-(β-D-Xylofuranosyl)-5-(2-chlorovinyl)cytosine,

[1325] 1-(β-D-Xylofuranosyl)-5-(2-bromovinyl)cytosine,

[1326] 1-(β-D-Xylofuranosyl)-5-(2-iodovinyl)cytosine,

[1327] 1-(β-D-Xylofuranosyl)-5-(2-methoxylcarbonyl vinyl)cytosine,

[1328] 3-(β-D-Xylofuranosyl)-5-(2-hydroxycarbonylvinyl)cytosine,

[1329] 1-(β-D-Xylofuranosyl)-5-phenylcytosine,

[1330] 1-(β-D-Xylofuranosyl)-5-benzylcytosine,

[1331] 1-(β-D-Xylofuranosyl)-2-chloroadenine,

[1332] 1-(β-D-Xylofuranosyl)-6-chloropurine,

[1333] 1-(β-D-Xylofuranosyl)-2,6-dichloropurine,

[1334] 1-(β-D-Xylofuranosyl)-2-acetamido-6-chloropurine,

[1335] 1-(β-D-Xylofuranosyl)-2-acetamido-6-methoxypurine,

[1336] 1-(β-D-Xylofuranosyl)-6-methoxypurine and

[1337] 1-(β-D-Xylofuranosyl)-6-methylmercaptopurine.

Example 39

[1338] 2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-N4-hydroxycytidine.

[1339] To a stirred solution of2′,3′-O-isopropylidene-5-O-triphenylmethyluridine (1 g) in 50 mL ofanhydrous acetonitrile and triethylamine (0.76 g) are added2,4,6-triisopropylbenzenesulfonyl chloride (1.15 g) and DMAP (232 mg) at0° C., and the reaction mixture is stirred for 1 day at roomtemperature. Hydroxylamine hydrochloride (263 mg) is then added, and themixture is further stirred for 1 day at room temperature. The reactionis quenched by addition of water, and the product is extracted withchloroform (200 mL). The organic layer is washed with brine, dried overMgSO₄, and concentrated in vacuo. The residue is purified by silica gelcolumn chromatography (5% MeOH in CHCl₃) to give2′,3′-O-isopropylidene-5′-O-trityl-N⁴-hydroxy-cytidine (723 mg, 70%) asa white solid. Mp: 99-101° C. ¹H NMR (CDCl₃) δ 1.34 (s, 3H), 1.56 (s,3H), 3.40-3.73 (m, 2H), 4.26 (br s, 1H), 4.79-4.81 (m, 2H), 5.34 (d,J=8.12 Hz, 1H), 5.88 (br s, 1H), 6.88 m(d, J=8.12 Hz, 1H), 7.22-7.41 (m,15H).

[1340] In a similar manner but using the corresponding 5-substituteduracil nucleosides, the followingN⁴-hydroxy-2′,3′-O-isopropylidene-5′-O-triphenylmethylcytidinederivatives are synthesized:

[1341]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-fluoro-N⁴-hydroxycytidine,

[1342]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-chloro-N⁴-hydroxycytidine,

[1343]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-bromo-N⁴-hydroxycytidine,

[1344]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-iodo-N⁴-hydroxycytidine,

[1345]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-methyl-N⁴-hydroxycytidine,

[1346]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-ethyl-N⁴-hydroxycytidine,

[1347] 2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-n-propyl-N⁴-hydroxycytidine,

[1348]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-i-propyl-N⁴-hydroxycytidine,

[1349]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-vinyl-N⁴-hydroxycytidine,

[1350] 22′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-ethynyl-N⁴-hydroxycytidine,

[1351] 2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-(2-chlorovinyl)-N⁴-hydroxycytidine,

[1352]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-(2-bromovinyl)-N⁴-hydroxycytidine,

[1353]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-(2-iodovinyl)-N⁴-hydroxycytidine,

[1354]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-(2-methoxycarbonylvinyl)-N⁴-hydroxycytidine,

[1355]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-(2-hydroxycarbonylvinyl)-N⁴-hydroxycytidine,

[1356]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-phenyl-N⁴-hydroxycytidineand

[1357]2′,3′-O-Isopropylidene-5′-O-triphenylmethyl-5-benzyl-N⁴-hydroxycytidine.

[1358] In a similar manner but using the corresponding 5-substituted2′,5′-di-O-acetyl-3′-deoxyuridines, the followingN4-hydroxy-2′,5′-di-O-acetyl-3′-deoxycytidine derivatives aresynthesized:

[1359]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-N⁴-hydroxycytosine,

[1360]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-fluoro-N⁴-hydroxycytosine,

[1361]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-chloro-N⁴-hydroxycytosine,

[1362]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-cyano-N4-hydroxycytosine,

[1363]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-ethoxycarbonyl-N⁴-hydroxycytosine,

[1364]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-aminocarbonyl-N⁴-hydroxycytosine,

[1365]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-acetyl-N⁴-hydroxycytosine,

[1366]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-methyl-N⁴-hydroxycytosine,

[1367]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-n-propyl-N⁴-hydroxycytosine,

[1368]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-n-propyl-N⁴-hydroxycytosine,

[1369]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-vinyl-N⁴-hydroxycytosine,

[1370]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-allyl-N⁴-hydroxycytosine,

[1371]5-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-allyl-N⁴-hydroxycytosine,

[1372]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-ethyovinyl)-N⁴-hydroxycytosine

[1373]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-bromovinyl)-N⁴-hydroxycytosine,

[1374]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-iodovinyl)-N⁴-hydroxycytosine,

[1375]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-methoxylcarbonylvinyl)-N⁴-hydroxycytosine,

[1376]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-(2-hydroxycarbonylvinyl)-N⁴-hydroxycytosine,

[1377]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-phenyl-N⁴-hydroxycytosineand

[1378]1-(2,5-Di-O-acetyl-3-deoxy-β-D-erythropentofuranosyl)-5-benzyl-N4-hydroxycytosine,

[1379] In a similar manner but using the corresponding 5-substituted3′,5′-di-O-acetyl-2′-deoxyuridines, the followingN⁴-hydroxy-3′,5′-di-O-acetyl-N⁴-hydroxy-2′-deoxycytidine derivatives aresynthesized:

[1380] 3′,5′-Di-O-acetyl-2′-deoxy-N⁴-hydroxycytidine,

[1381] 3′,5′-Di-O-acetyl-2′-deoxy-5-fluoro-N⁴-hydroxycytidine,

[1382] 3′,5′-D 1-O-acetyl-2′-deoxy-5-chloro-N⁴-hydroxycytidine,

[1383] 3′,5′-Di-O-acetyl-2′-deoxy-5-bromo-N⁴-hydroxycytidine,

[1384] 3′,5′-Di-O-acetyl-2′-deoxy-5-iodo-N⁴-hydroxycytosine,

[1385] 3′,5′-Di-O-acetyl-2′-deoxy-5-cyano-N⁴-hydroxycytidine,

[1386] 3′,5′-Di-O-acetyl-2′-deoxy-5-ethoxycarbonyl-N⁴-hydroxycytidine,

[1387] 3′,5′-Di-O-acetyl-2′-deoxy-5-aminocarbonyl-N⁴-hydroxycytidine,

[1388] 3′,5′-Di-O-acetyl-2′-deoxy-5-acetyl-N⁴-hydroxycytidine,

[1389] 3′,5′-Di-O-acetyl-2′-deoxy-5-methyl-N⁴-hydroxycytidine,

[1390] 3′,5′-Di-O-acetyl-2′-deoxy-5-ethyl-N⁴-hydroxycytosine,

[1391] 3′, 5′-Di-O-acetyl-2′-deoxy-5-n-propyl-N⁴-hydroxycytidine,

[1392] 3′, 5′-Di-O-acetyl-2′-deoxy-5-i-propyl-N⁴-hydroxycytidine,

[1393] 3′, 5′-Di-O-acetyl-2′-deoxy-5-vinyl-N⁴-hydroxycytidine,

[1394] 3′,5′-Di-O-acetyl-2′-deoxy-5-allyl-N⁴-hydroxycytidine,

[1395] 3′, 5′-Di-O-acetyl-2′-deoxy-5-ethynyl-N⁴-hydroxycytidine,

[1396] 3′,5′-Di-O-acetyl-2′-deoxy-5-(2-chlorovinyl)-N⁴-hydroxycytidine,

[1397] 3′,5′-Di-O-acetyl-2′-deoxy-5-(2-bromovinyl)-N⁴-hydroxycytidine,

[1398] 3′,5′-Di-O-acetyl-2′-deoxy-5-(2-iodovinyl)-N⁴-hydroxycytidine,

[1399]3′,5′-Di-O-acetyl-2′-deoxy-5-(2-methoxylcarbonylvinyl)-N⁴-hydroxycytidine,

[1400]3′,5′-Di-O-acetyl-2′-deoxy-5-(2-hydroxycarbonylvinyl)-N⁴-hydroxycytosine,

[1401] 3′,5′-Di-O-acetyl-2′-deoxy-5-phenyl-N⁴-hydroxycytidine and3′,5′-Di-O-acetyl-2′-deoxy-5-benzyl-N⁴-hydroxycytidine.

Example 40

[1402] N⁴-Hydroxycytidine.

[1403] 2′,3′-O-Isopropylidene-5′-O-trityl-N⁴-hydroxycytidine (500 mg,0.92 mmol) is dissolved in 50 mL of a mixture of trifluoroacetic acidand water (2:1, v/v), and the solution is stirred for 3 h at 50° C.After cooling to room temperature, the solvent is removed by evaporationand coevaporated with ethanol (3×20 mL). The residue is purified bysilica gel column chromatography (20% MeOH in CHCl₃) to giveN⁴-hydroxycytidine (215 mg) as a white solid which is recrystallizedfrom hot ethanol; mp. 173-176° C. ¹H NMR (DMSO-d₆) δ 3.66-3.71 (m, 2H),3.93 (br s, 1H), 4.08-4.15 (m, 2H), 5.17-5.23 (m, 2H, D₂O exchangeable),5.43 (d, J=6.00 Hz, 1H, D₂O exchangeable), 5.73 (d, J=8.16 Hz, 1H), 5.90(d, J=8.12 Hz, 1H), 7.28 (d, J=8.40 Hz, 1H), 9.65 (s, 1H, D₂Oexchangeable), 10.15 (s, 1H, D₂0 exchangeable). Anal. Calcd forC9H13N3O6: C, 41.70; H, 5.05; N, 16.21. Found: C, 41.85; H, 5.14; N,16.34.

[1404] In a similar manner but using the corresponding 5-substituted2′,3′-O-isopropylidene-5-O-triphenylmethyl-N⁴-hydroxycytidinenucleosides, the following N⁴-hydroxy-5-substituted cytidine aresynthesized:

[1405] 5-Fluoro-N⁴-hydroxycytidine,

[1406] 5-Chloro-N⁴-hydroxycytidine,

[1407] 5-Bromo-N⁴-hydroxycytidine,

[1408] 5-Iodo-N⁴-hydroxycytidine,

[1409] 5-Methyl-N⁴-hydroxycytidine,

[1410] 5-Ethyl-N⁴-hydroxycytidine,

[1411] 5-n-Propyl-N⁴-hydroxycytidine,

[1412] 5-i-Propyl-N⁴-hydroxycytidine,

[1413] 5-Vinyl-N⁴-hydroxycytidine,

[1414] 5-Ethynyl-N⁴-hydroxycytidine,

[1415] 5-(2-chlorovinyl)-N⁴-hydroxycytidine,

[1416] 5-(2-bromovinyl)-N⁴-hydroxycytidine,

[1417] 5-(2-iodovinyl)-N⁴-hydroxycytidine,

[1418] 5-(2-methoxycarbonylvinyl)-N⁴-hydroxycytidine,

[1419] 5-(2-hydroxycarbonylvinyl)-N⁴-hydroxycytidine,

[1420] 5-phenyl-N⁴-hydroxycytidine and

[1421] 5-benzyl-N⁴-hydroxycytidine.

[1422] In a similar manner but using methanolic ammonia instead oftrifluoroacetic acid, and the corresponding 5-substituted1-(2,5-di-O-acetyl-3-deoxy-β-D-erythro-pento-furanosyl)-N⁴-hydroxycytosinenucleosides, the following N⁴-hydroxy-5-substituted 3′-deoxycytidine aresynthesized:

[1423] 5-Fluoro-3′-deoxy-N⁴-hydroxycytidine,

[1424] 5-Chloro-3′-deoxy-N⁴-hydroxycytidine,

[1425] 5-Bromo-3′-deoxy-N⁴-hydroxycytidine,

[1426] 5-Iodo-3′-deoxy-N⁴-hydroxycytidine,

[1427] 5-Methyl-3′-deoxy-N⁴-hydroxycytidine,

[1428] 5-Ethyl-3′-deoxy-N⁴-hydroxycytidine,

[1429] 5-n-Propyl-3′-deoxy-N⁴-hydroxycytidine,

[1430] 5-i-Propyl-3′-deoxy-N⁴-hydroxycytidine,

[1431] 5-Vinyl-3′-deoxy-N⁴-hydroxycytidine,

[1432] 5-Ethynyl-3′-deoxy-N⁴-hydroxycytidine,

[1433] 5-(2-chlorovinyl)-3′-deoxy-N⁴-hydroxycytidine,

[1434] 5-(2-bromovinyl)-3′-deoxy-N⁴-hydroxycytidine,

[1435] 5-(2-iodovinyl)-3′-deoxy-N⁴-hydroxycytidine,

[1436] 5-(2-methoxycarbonylnylyl)-3′-deoxy-N⁴-hydroxycytidine,

[1437] 5-(2-hydroxycarbonylvinyl)-3′-deoxy-N⁴-hydroxycytidine,

[1438] 5-phenyl-3′-deoxy-N⁴-hydroxycytidine and

[1439] 5-benzyl-3′-deoxy-N⁴-hydroxycytidine.

[1440] In a similar manner but using methanolic ammonia instead oftrifluoroacetic acid, and the corresponding 5-substituted3′,5′-di-O-acetyl-2′-deoxy-N4-hydroxycytosine nucleosides, the followingN⁴-hydroxy-5-substituted 2′-deoxycytidine are synthesized:

[1441] 5-Fluoro-2′-deoxy-N⁴-hydroxycytidine,

[1442] 5-Chloro-2′-deoxy-N⁴-hydroxycytidine,

[1443] 5-Bromo-2′-deoxy-N⁴-hydroxycytidine,

[1444] 5-Iodo-2′-deoxy-N⁴-hydroxycytidine,

[1445] 5-Methyl-2′-deoxy-N⁴-hydroxycytidine,

[1446] 5-Ethyl-2′-deoxy-N⁴-hydroxycytidine,

[1447] 5-n-Propyl-2′-deoxy-N⁴-hydroxycytidine,

[1448] 5-i-Propyl-2′-deoxy-N⁴-hydroxycytidine,

[1449] 5(2-Vinyl2′β-deoxy-N⁴-hydroxycytidine,

[1450] 5-E Vinyl-2′-deoxy-N4-hydroxycytidine,

[1451] 5-(2-iodovinyl)-2′-deoxy-N⁴-hydroxycytidine,

[1452] 5-(2-bromovinyl)-2′-deoxy-N⁴-hydroxycytidine,

[1453] 5-(2-iodovinyl)-2′-deoxy-N⁴-hydroxycytidine,

[1454] 2,5-(2-methoxycarbonylvinyl)-2′′-deoxy-N⁴-hydroxycytidine,

[1455] 5-(2-hydroxycarbonylvinyl)-2′-deoxy-N⁴-hydroxycytidine,

[1456] 5-phenyl-2′-deoxy-N⁴-hydroxycytidine and

[1457] 5-benzyl-2′-deoxy-N⁴-hydroxycytidine.

Example 41

[1458]2,3′-Anhydro-1-(2-deoxy-2-fluoro-5-O-trityl-)-D-lyxofuranosyl)thymine(194, R=Tr).

[1459] A solution of1-(2-deoxy-2-fluoro-3-O-mesyl-5-O-triphenylmethyl-β-D-arabino-furanosyl)thymine(193, R=Tr, 6.0 g) and DBU (3.0 mL) in methylene chloride (50 mL) isheated at reflux for 16 hours. After concentration of the mixture invacuo, the residue is chromatographed on a silica gel column usingchloroform as the eluent to give 4.4 g of2,3′-anhydro-1-(2′-deoxy-2′-fluoro-5-O-trityl-β-D-lyxofuranosyl)thymine(194, R=Tr), mp 252-255° C. after recrystallization from methanol. ¹HNMR (DMSO-d₆); δ 1.80 (s, 3H, Me), 4.61 (1H, m), 5.40 (dm, 1H), 5.89(1H, ddd), 5.96 (1H, dd, H-1′), 7.30 (15H, Tr), 7.66 (s, 1H, H-6).

Example 42

[1460]1-(2,3-Dideoxy-2′fluoro-5′-O-trityl-βD-glycero-pento-2-enofuranosyl)-thymine(195, R=Tr).

[1461] A suspension of 194 (646 mg) and t-BuOK (270 mg) in DMSO (10 mL)is stirred at room temperature for 2 hours and then filtered. Thefiltrate is concentrated in vacuo and the residue is chromatographed ona silica gel column (CHCl₃/MeOH, 49:1 v/v) to give 600 mg of 195, mp.176-180° C. (from EtOH). ¹H NMR (DMSO-d₆) δ 1.27 (s, 3H, Me), 3.21 (m,2H, H-5,5″), 4.98 (m, 1H, H-4′), 6.17 (t, 1H, H-1′, J1′,2′=J1′,F=1.5Hz), 6.81 (m, IH, H-3′), 7.32 (m, 16H, H-6, Tr), 11.52 (s, 1H, NHexchangeable).

Example 43

[1462] 1-(2,3-Dideoxy-2-fluoro-β-D-glycero-2-enofuranosyl)thymine (196).

[1463] A solution of 195 (600 mg) in 80% aqueous acetic acid (10 mL) isheated under reflux for 20 minutes and then concentrated to dryness invacuo. The residue is chromatographed on a column of silica gel(CHCl₃/MeOH, 9:1 v/v) to give 100 mg of 196, mp 154-159° C. (fromEtOH-H₂O ). ¹H NMR (DMSO-d₆) δ 1.76 (s, 3H, Me), 3.61 (m, 2H, H-5′,5″),4.79 (m, 1H, H-4′), 5.15 (t, 1H, 5′-OH, exchangeable), 5.99 (m, 1H,H-1′), 6.76 (m, 1H, H-3′), 7.88 (s, 1H, H-6), 11.43 (s, 1H, NH,exchangeable).

Example 44

[1464] (1S,2S, 3R,4R)-4-(tert-Butoxymethyl)-2,3-(isopropylidenedioxy)cyclopentan-1-ol(219).

[1465] To a solution of 4-(t-butoxymethyl)cyclopentane-2,3-diol (218, 5g) and CeCl₃ 7H₂O (7.69 g) in methanol (80 mL) is added NaBH₄ (1.01 g)at 0° C., and the mixture is stirred for 1 hour at 0° C. The reaction isquenched by addition of cold water, and extracted with ethyl acetate(2×300 mL). The combined organic extracts are washed with brine (2×200mL), dried over Na2SO4, and then concentrated in vacuo. The residue ischromatographed on a silica gel column (30% ethyl acetate in n-hexane)to give 219 (4.8 g, 95%) as a syrup. ¹H-NMR (CDCl₃) δ 1.13 (s, 9H,t-Bu), 1.34 (s, 3H, Me), 1.48 (s, 3H, Me), 1.83 (m, 2H, 5a,b-H), 2.19(m, 1H, 4-H), 2.44 (d, OH, exchangeable), 3.20 (dd, J=4.5, 8.8 Hz, 1H,6a-H), 3.31 (dd, J=4.5, 8.8 Hz, 1H, 6b-H), 4.23 (m, 1H, 1-H), 4.44 (m,2H, 2-H, 3-H). Anal. Calcd for C₁₃H₂₄O₄: C, 63.91; H, 9.90. Found: C,64.09; H, 9.87.

Example 45

[1466] (1S, 2S, 3R,4R)-4-(tert-Butoxymethyl)-2,3-(isopropylidenedioxy)-1-mesyloxycyclopentane(220).

[1467] To a solution of 219 (6.50 g) and triethylamine (7.3 g) inmethylene chloride (170 mL) is added mesyl chloride (4.73 g) dropwise at0° C. After 45 minutes, water (270 mL) is added. The aqueous layer isextracted with methylene chloride (3×200 mL). The organic layers arecombined, washed with brine (2×200 mL), dried over Na₂SO₄, andconcentrated in vacuo to give crude 220, which is sufficiently pure tobe used directly in the next step.

Example 46

[1468] (1R, 2S, 3R,4R)-1-Azido-4-(tert-butoxymethyl)-2,3-(isopropylidenedioxy)cyclopentane(221).

[1469] A mixture of 220 obtained above and sodium azide (17.3 g) in DMF(300 mL) is heated at 140° C. overnight with stirring. The mixture isfiltered and the filtrate is concentrated in vacuo. The residue ispartitioned between ethyl acetate (150 mL) and water (50 mL). Theorganic layer is dried over Na₂SO₄, concentrated in vacuo, and theresidue is chromatographed on a silica gel column (1-4% gradient, ethylacetate in n-hexane) to give 221 (5.9 g) as an oil. ¹H NMR (CDCl₃) δ1.18 (s, 9H, t-Bu), 1.30 (s, 3H, Me), 1.46 (s, 3H, Me), 1.71 (m, 1H,5a-H), 2.29 (m, 2H, 4-H, 5b-H), 3.29 (dd, J=6.7, 8.8 Hz, 1H, 6a-H), 3.37(dd, J=7.0, 8.8 Hz, 1H, 6b-H), 3.96 (m, 1H, 1-H), 4.40 (dd, J=2.3, 6.1Hz, 1H, 3-H), 4.48 (dd, J=2.0, 6.1 Hz, 1H, 2-H). Anal. Calcd forC₁₃H₂₃N₃O₃0.13EtOAc: C, 57.95; H, 8.65, N, 14.99. Found: C, 58.25; H,8.71; N, 14.76.

Example 47

[1470] (1R, 2S, 3R,4R)-4-(tert-Butoxymethyl)-2,3-(isopropylidenedioxy)-1-cyclopentylamine(222).

[1471] A suspension of 221 (4.0 g) and 10% Pd/C (1.0 g) in anhydrousethanol (140 mL) is shaken under 20 psi of H₂ for 5 hours. The mixtureis filtered, and the filtrate is concentrated in vacuo to give crude 222(3.6 g, quantitative), which is used directly in the next step withoutfurther purification. ¹H NMR (CDCl₃) δ 1.18 (s, 9H, t-Bu), 1.28 (s, 3H,Me), 1.36 (m, 1H, 5a-H), 1.45 (s, 3H, Me), 1.89 (br s, 2H, NH₂),2.24-2.36 (m, 2H, 4-H, 5b-H), 3.34-3.43 (m, 3H, 1-H, 6a,b-H), 4.21 (dd,J=2.6, 6.2 Hz, 1H, 3-H), 4.48 (dd, J=2.8, 6.2 Hz, 1H, 2-H). Anal. Calcdfor C₁₃H₂₆NO₃0.16H₂O : C, 63.41; H, 10.37, N, 5.69. Found: C, 63.09; H,10.16; N, 5.59.

Example 48

[1472]N-{[1R,2S,3R,4R)-4-(tert-Butoxymethyl)-2,3-(isopropylidenedioxy)cyclopentyl]-aminocarbonyl}-3-methoxy-2-propenamide(223).

[1473] A mixture of silver cyanate (7.60 g, dried in vacuo overphosphorus pentoxide in the dark at 100° C. for 3 hours),β-methoxyacryloyl chloride (2.64 g) in anhydrous benzene (30 mL) isheated under reflux for 30 minutes, and then is allowed to cool to roomtemperature. After precipitation is settled, 22.5 mL of the supernatant,which contains β-methoxyacryloyl isocyanate) is added during 15 minutesto a solution of 222 (3.0 g) in dry DMF (50 mL) at −15 to −20° C. undernitrogen. The mixture is stirred for 2 hours at −15° C. and then 10 morehours at room temperature under nitrogen. After concentration in vacuoand coevaporation with toluene (2×20 mL), the product 223 solidifies(4.0 g). ¹H NMR (CDCl₃) δ 1.17 (s, 9H, t-Bu), 1.28 (s, 3H, Me), 1.47 (s,3H, Me), 1.58 (m, 1H, 5′a-H), 2.28 (m, 1H, 4-H), 2.36-2.43 (m, 1H,5′b-H), 3.33-3.42 (m, 2H, 6′a,b-H), 3.73 (s, 3H, OMe), 4.20 (m, 1H,3′-H), 4.45 (m, 2H, 1′-H, 2′-H), 5.35 (d, J 12.3 Hz, 1H, 5-H), 7.67 (d,J 12.3 Hz, 1H, 6-H), 8.72 (br s, 1H, NH), 9.35 (br s, 1H, NH). Anal.Calcd for C₁₈H₃₀N₂O_(6′): C, 58.36; H, 8.16, N, 7.56. Found: C, 58.28;H, 8.16; N, 7.60.

Example 49

[1474] (1′R, 2 S, 3R, 4R)-1-[4-(tert-Butoxymethyl)-2,3-isopropylidenedioxy)cyclopentan-1-yl]uracil(5′-tert-Butyl-2,3′-O-isopropylidene-carba-uridine, 224).

[1475] A solution of 223 (4.2 g) in ethanol (25 mL) and ammoniumhydroxide (30% 11 mL) is heated at 100° C. in a steel bomb for 12 hours.After removal of the solvents, the residue is chromato graphed over asilica gel column (ethylacetate-n-hexane, 1:1 v/v) to give 224 (3.21 g)as a white foam. UV (MeOH) kλ_(max) 266.0 nmn. ¹H NMR (CDCl₃) δ 1.19 (s,9H, t-Bu), 1.30 (s, 3H, Me), 1.54 (s, 3H, Me), 1.97 (mn, 1H, 5′a-H),2.32-2.41 (mn, 2H, 4′-H, 5′b-H), 3.43-3.50 (mn, 2H, 6′a,b-H), 4.48 (dd,J=4.1, 6.5 Hz, 1H, 3′-H), 4.65-4.75 (mn, 2H, 1′-H, 2′-H), 5.72 (d, J=8.0Hz, 1H, 5-H), 7.35 (d, J=8.0 Hz, 1H, 6-H), 8.63 (br s, 1H, NH). Anal.Calcd for C₁₇H₂₆N₂O₅: C, 60.34; H, 7.74, N, 8.28. Found: C, 60.06; H,7.70; N, 8.14.

Example 50

[1476] (1′R,2 ′S,3 ′R,4′R)-1-[4-(tert-Butoxymethyl)-2,3-isopropylidenedioxy)cyclopentan-1-yl]-5-fluorouracil(5′-O-tert-Butyl-2 , 3′-O-isopropylidene-carba-5-fluorouridine, 225).

[1477] A fluorine-nitrogen mixture containing 5% of fluorine is bubbledcarefully into a solution of 224 (2.50 g) in acetic acid (600 mL) for 30minutes at room temperature. The mixture is stirred until no UVabsorption is detected on TLC plate. The solvent is removed in vacuo,and the residue is coevaporated with acetic acid (20 mL) to dryness. Theresidue is treated with triethylamine for 1.5 hours at 50° C., and thenconcentrated in vacuo to dryness. The residue is purified by silica gelcolumn chromatography (ethylacetate-n-hexane, 1:1 v/v) to give 225 (1.31g) as a white foam. TV (MeOH) λ_(max) 271.5 nm. ¹H NMR (CDCl₃) δ 1.22(s, 9H, t-Bu), 1.31 (s, 3H, Me), 1.55 (s, 3H, Me), 1.85 (m, 1H, 5′a-H),2.38-2.51 (m, 2H, 4′-H, 5′b-H), 3.44-3.52 (m, 2H, 6′a,b-H), 4.47 (dd,J=3.4, 6.2 Hz, 1H, 3′-H), 4.58 (t, J=6.0 Hz, 1H, 1′-H), 4.87 (dd, J=8.9,14.5 Hz, 1H, 2′-H), 7.61 (d, J=6.1 Hz, 1H, 6-H), 8.77 (br s, 1H, NH).Anal. Calcd for C₁₇H₂₅FN₂O₅.0.25H₂O : C, 56.58; H, 7.12, N, 7.76. Found:C, 56.20; H, 7.02; N, 7.50.

Example 51

[1478] (1′R, 2′S, 3′R,4′R)-1-[4-(tert-Butoxymethyl)-2,3-isopropylidenedioxy)cyclopentan-1-yl]-5-fluorocytosine(226). (5′-O-tert-Butyl-2′,3′-O-isopropylidene-carba-5-fluorocytidine)

[1479] A mixture of 225 (350 mg), triethylamine (190 mg),2,4,6-triisopropylbenzenesulfonyl chloride (590 mg) and DMAP (230 mg) inacetonitrile (50 mL) is stirred for 1 day at room temperature. Ammoniumhydroxide solution (30%, 15 mL) is added, and the mixture is furtherstirred 5 hours. The reaction is quenched by addition of chloroform (250mL) and water (10 mL). The organic layer is washed with brine, driedover Na₂SO₄, and concentrated in vacuo. The residue is purified bysilica gel column chromatography (5% MeOH in CHCl₃, v/v) to give 226(205 mg), mp 128-130° C. UV (MeOH) λ_(max) 286.5 nm. ¹H NMR (CDCl₃) δ1.19 (s, 9H, t-Bu), 1.29 (s, 3H, Me), 1.53 (s, 3H, Me), 2.02 (dt,J=10.2, 12.8 Hz, 1H, 5′a-H), 2.32 (m, 1H, 4′-H), 2.42 (dt, J=8.0, 12.7Hz, 1H, 5′b-H), 3.42 (dd, J=6.1, 8.7 Hz, 1H, 6′a-H), 3.52 (dd, J=4.1,8.8 Hz, 1H, 6′b-H), 4.49 (dd, J=5.1, 6.3 Hz, 1H, 3′-H), 4.60 (m, 1H,1′-H), 4.79 (dd, J=5.0, 6.4 Hz, 1H, 2′-H), 7.49 (d, J 6.1 Hz, 1H, 6-H).HR-FAB MS Obsd; m/z 356.1981. Calcd for C₁₇H₂₆FN₃O₄: m/z 356.1986 (M+1)⁺.

Example 52

[1480] (1′R,2 ′S, 3 ′R,4′R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)cyclopentan-1-yl]-5-fluorocytosine(carba-5-fluorocytidine, 227).

[1481] A solution of 226 (180 mg) in a 2:1 (v/v) mixture oftrifluoroacetic acid and water (40 mL) is stirred for 3 hours at 50° C.The solvents are removed in vacuo, and the residue is coevaporated withethanol (2×30 mL), and purified on a silica gel column (MeOH-CHCl3, 1:5v/v) to give 227 (47.5 mg) as a foam. WV (H₂O ) λ_(max) 284 nm (s 5,876,pH 7), 293.5 nm (ε 7,440, pH 2), 284 5 nm (ε 5,883, pH 11). ¹H NMR(DMSO-d₆) δ 1.19 (m, 1H, 5′a-H), 1.92 (m, 1H, 4′a-H), 2.00 (ddd, J=8.3,8.7, 12.5 Hz, 1H, 5′b-H), 3.42 (m, 2H, 6′ab-H), 3.70 (dd, J=2.9, 5.3 Hz,1H, 3′b-H), 3.98 (dd, J=5.2, 9.0 Hz, 1H, 2′-H), 4.10 (d, J=4.5, 1H, OH,exchangeable), 4.51 (br s, 1H, OH, exchangeable), 4.60 (dd, J=9.0, 18.2Hz, 1H, 1′-H), 4.73 (d, J=6.1 Hz, 1H, OH, exchangeable), 7.33 (bs, 1H,exchangeable), 7.55 (bs, 1H, exchangeable), 7.98 (d, J=7.3 Hz, 1H, 6-H).HR-FAB MS Obsd; m/z 260.1054. Calcd for C₁₇H₂₆FN₃O₄: m/z 260.1047(M+1)⁺.

[1482] In a similar manner but using the corresponding 5-substitutedderivatives, the following 5-substituted carba-nucleosides are prepared:-Chloro-carba-uridine,

[1483] 5-Bromo-carba-uridine,

[1484] 5-Bodo-carba-uridine,

[1485] 5-Cyano-carba-uridine,

[1486] cara-Uridine-5-carboxylic acid,

[1487] 5-Ethoxycarbonyl-carba-uridine,

[1488] carba-Uridine-5-carboxamide,

[1489] 5-Hydroxymethyl-carba-uridine,

[1490] 5-Nitro-carba-uridine,

[1491] 5-Amino-carba-uridine

[1492] 5-Chloro-carba-cytidine,

[1493] 5-Bromo-carba-cytidine,

[1494] 5-lodo-carba-cytidine,

[1495] 5-Cyano-carba-cytidine,

[1496] cara-Cytidine-5-carboxylic acid,

[1497] 5-Ethoxycarbonyl-carba-cytidine,

[1498] carba-Cytidine-5-carboxamide,

[1499] 5-Hydroxymethyl-carba-cytidine,

[1500] 5-Nitro-carba-cytidine and

[1501] 5-Amino-carba-cytidine.

[1502] XI. Biological Methods

[1503] This invention further provides an efficient process to quantifythe viral load in a host using quantitative real-timereverse-transcription polymerase chain reaction (“Q-RT-PCR”). Theprocess involves using a quenched fluorescent probe molecule that can behybridized to a target viral DNA or RNA. Therefore, upon exonucleolyticdegradation, a detectable fluorescent signal can be monitored.Therefore, the RT-PCR amplified DNA or RNA can be detected in real timeby monitoring the presence of fluorescence signals.

[1504] In a specific embodiment of the invention, the use of RT-PCR toquantitate the viral load of a Flaviviridae virus is provided.

[1505] In a more specific embodiment, the use of RT-PCR to quantitatethe viral load of BVDV in a MDBK cell line or a host sample is provided.

[1506] In a further embodiment of the invention, a probe moleculedesigned to fluoresce upon exonucleolytic degradation and to becomplementary to the BVDV NADL NS5B region is provided.

[1507] In a more specific embodiment of the invention, a probe moleculewith a sequence of 5′,6-fam-AAATCCTCCTAACAAGCGGGTTCCAGG-tamara 3′(Sequence ID No 1) and primers with a sequence of sense:5′-AGCCTTCAGTTTCTTGCTGATGT-3′(Sequence ID No 2) and antisense:5′-TGTTGCGAAAGCACCAACAG-3′ (Sequence ID No 3) is provided.

[1508] In a specific embodiment of the invention, the use of RT-PCR toquantitate viral load of HCV in a host derived sample or a cell line inreal time is provided;

[1509] In a more specific embodiment of the invention, the use ofRT-PCR, a probe molecule designed to fluoresce upon exonucleolyticdegradation and to be complementary to the HCV genome is provided In amore specific embodiment of the invention, the use of RT-PCR, a probemolecule designed to fluoresce upon exonucleolytic degradation and to becomplementary to the HCV 5′ untranslated region is provided In a morespecific embodiment of the invention, a probe molecule with a sequenceof 5′,6-fam-CCTCCAGGACCCCCCCTCCC-tamara 3′ (Sequence ID No 4) andprimers with a sequence of sense: 5′-AGCCATGGCGTTAGTA(T/C)GAGTGT-3′(Sequence ID No 5) and antisense: 5′-TTCCGCAGACCACTATGG-3′ (Sequence IDNo 6) is provided.

[1510] A. RNA Isolation and Quantitative RT-PCR Analysis

[1511] An effective process to quantify the viral load in a host, termedreal-time polymerase chain reaction (“RT-PCR”) is provided. The processinvolves using a quenched fluorescent probe molecule that can behybridized to viral DNA or RNA. Therefore, upon exonucleolyticdegradation, a detectable fluorescent signal can be monitored.Therefore, the RT-PCR amplified DNA or RNA is detected in real time bymonitoring the presence of fluorescence signals.

[1512] As one illustration of this method, in the case of BVDV in MDBKcells, in a first step, viral RNA is isolated from 140 μL of the cellculture supernatant by means of a commercially available column (ViralRNA extraction kit, QiaGen, Calif.). The viral RNA is then eluted fromthe column to yield a total volume of 60 μL, and subsequently amplifiedwith a quantitative RT-PCR protocol using a suitable primer for the BVDVNADL strain. A quenched fluorescent probe molecule is hybridized to theBVDV DNA, which then undergoes exonucleolytic degradation resulting in adetectable fluorescent signal. Therefore, the RT-PCR amplified DNA wasdetected in real time by monitoring the presence of fluorescencesignals. The TaqMan probe molecule(5′-6-fam-AAATCCTCCTAACAAGCGGGTTCCAGG-tamara 3′ [Sequence ID No 1] andprimers (sense: 5′-AGCCTTCAGTTTCTTGCTGATGT-3′ [Sequence ID No 2]; andantisense: 5′-TGTTGCGAAAGCACCAACAG-3′ [Sequence ID No 3]) were designedwith the aid of the Primer Express software (PE-Applied Biosystems) tobe complementary to the BVDV NADL NS5B region. A total of 10 μL of RNAwas analyzed in a 50 μL RT-PCR mixture. Reagents and conditions used inquantitative PCR were purchased from PE-Applied Biosystems. The standardcurve that was created using the undiluted inoculum virus ranged from6000 plaque forming units (PFU) to 0.6 PFU per RT-PCR mixture. A linearrange of over 4-logs was routinely obtained.

[1513] A comparable approach can be taken to measure the amount of otherFlaviviridae (more importantly HCV, YFV, Dengue, West Nile Virus andothers) in a clinical sample or in a tissue culture sample. For example,the combination of HCV RNA purification with real-time RT-PCR using thefollowing primers (5′-TTCCGCAGACCACTATGG-3′ [Sequence ID No. 4] and5′-AGCCATGGCGTTAGTATGAGTGT-3′ [Sequence ID No. 5]) and probe(5′-6-fam-CCTCCAGGACCCCCCCTCCC-tamara-3′ [Sequence ID No. 6]) resultedin a 7-log linear range of viral load detection.

[1514] B. Cell/Viral Materials

[1515] One of the best characterized members of the Pestivirus genus isBVDV. BVDV and HCV share at least three common features, which are thefollowing: (1) they both undergo IRES-mediated translation; (2) NS4Acofactor is required by their NS3 serine protease; and (3) they undergosimilar polyprotein processing within the non-structural region,especially at the NS5A and NS5B junction site.

[1516] The BVDV replication system was used for the discovery ofanti-Flaviviridae compounds. The compounds described herein are activeagainst Pestiviruses, Hepaciviruses and/or Flaviviruses.

[1517] Maldin-Darby bovine kidney (MDBK) cells were grown and maintainedin a modified eagle medium (DMEM/F12; GibcoBRL), supplemented with 10%heat inactivated horse serum at 37° C. in a humidified, 5% CO₂,incubator.

[1518] Bovine viral diarrhea virus (BVDV), strain NADL, causes acytopathogenic effect (CPE) after infection of these cells.

[1519] C. Antiviral Assay

[1520] MDBK-cells, grown in DMEM/F12-10% horse serum (HS), were isolatedin standard techniques using trypsin-EDTA. Cells were seeded in a96-well plate at 5×10⁴ cells/well, with test compound (20 micromolar(μM) concentration) to give a total volume of 100 microliters (μL).After one hour, the media was removed and the cells were infected at amultiplicity of infection (MOI) of 0.02 or 0.002 in a total volume of 50μL for 45 minutes. Thereafter, the virus was removed and the cells werewashed twice with 100 μL of assay media. Finally, the infected cellswere incubated in a total volume of 100 μL containing the test compoundat 10, 40 or 100 μM concentration. After 22 hours, the cell supernatantwas collected by removing the cellular debris by low-speedcentrifugation, and subsequently tested for the presence of virus in aquantitative manner.

[1521] D. Cytotoxicity Testing of Anti-Flaviviridae Compounds

[1522] The cytotoxicity testing as performed here is a standardtechnique. Briefly, cells are seeded in 96-well plates at variousconcentrations (dependent on cell type, duration of assay), typically at5×10³ cells per well, in the presence of increasing concentrations ofthe test compound (0, 1, 3, 10, 33, and 100 μM). After a threeday-incubation, cell viability and mitochondrial activity are measuredby adding the MTS-dye (Promega), followed by a 3 hours incubation.Afterwards the plates containing the dye are read at 490 nm. Suchmethodologies are well described and available from the manufacturer(Promega).

Example 53

[1523] The BVDVRT-PCR Quantification Standard Curve

[1524] The standard BVDV virus stock contained 2×10⁶ PFU/mL, asdetermined by routine plaque assay (Mendez, E. et al. J. Virol. 1998,72, 4737). Viral RNA was extracted from 140 μL of this inoculum materialand eluted from a column using 60 μL of an elution buffer. This purifiedRNA material then was diluted stepwise from 10⁻¹ to 10⁻⁵. Using thereal-time RT-PCR amplification technique, 10 μL of each dilution wastested. The results of this dilution series are plotted in FIG. 1,relating PFU to concentration of standard. From this experiment, it isclear that this technology allows for reliable quantification over4-logs of virus (from 6000 to 0.6 PFU/input in amplification mix). Thelower limit of detection in this experiment is 0.6 PFU or −0.22 log PFU.Therefore, the real-time RT-PCR quantification values of test samplesbelow this detection limit were considered non-reliable.

Example 54

[1525] The B VD V Replication Cycle in MDBK Cells

[1526] In order to measure the BVDV production in MDBK cells and todetermine the optimal harvesting time over a certain period of time,cells were seeded at 5×10⁴ cells/well and infected either with MOI=0.02or MOI=0.002. After infection, the inoculum was removed and the cellswere washed twice with culture medium. At different time points, thecell supernatant was harvested; and, the amount of virus was measuredand compared to the original inoculum and the cell wash. At least 2wash-steps were needed to remove the inoculum virus, as shown in FIG. 2.The amount of virus produced 22 hours after infection approximatelyequals the amount of virus used to inoculate the cells. Based on theseresults, the time required for one replication cycle of BVDV in MDBKcells was 22 hours. Note that the detection level set in theseexperiments was based on the lower limit of detection as determined bythe standard curve.

Example 55

[1527] Evaluation of Antiviral Compounds Using RT-PCR

[1528] MDBK cells were seeded at 5×10⁴ cells/ well, infected with BVDVwith a multiplicity of infection (MOI) equal to 0.02 and grown for 22hours in the presence of a test compound. Cells that were not treatedwith a test compound were considered a negative control, while ribavirinserved as a positive control. Viral RNA was extracted and analyzed byreal time RT-PCR. A typical experiment, shown in FIG. 3, demonstratesthat the negative control and the majority of the treated cells producedcomparable amounts of virus (between 1.5 and 2 log PFU/input),effectively showing the test compounds as non-active. However, the cellstreated with the positive control, ribavirin (RIB) or with5-hydroxyuridine (β-D-CL) show an almost complete absence of viral RNA.RIB and β-D-CL reduce viral production by approximately 2 log PFU, or99%, in the 22 hour reproduction period. The exact potency of thesecompounds cannot be deduced from this kind of experiment, since thedetection limit in this experiment is set at −0.22 log PFU and only onecycle of viral replication occurs under the stated experimentalconditions.

[1529] Potencies, or the effect concentration of compounds that inhibitsvirus production by 50% or 90% (EC₅₀ or EC₉₀ values, respectively), ofanti-BVDV compounds were determined in a similar set of experiments, butover a broad range of test compound concentrations (0, 1, 3, 10, 33, 100μM). The EC₉₀ value refers to the concentration necessary to obtain a1-log reduction in viral production within a 22 hour period. Compoundsthat showed potent antiviral activity are listed in Table 21. This tablegives the maximal viral load reduction observed at a given concentration22 hours post infection. TABLE 21 BVDV viral load 22 hours postinfection ID n conc. (μM) Ave. Log Reduction β-D-AA 4 100 2.43 β-D-AI 3100 1.52 β-D-AJ 3 100 1.34 β-D-AK 4 100 1.90 β-D-AL 3 100 1.55 β-D-AN 2100 1.21 β-D-AO 2 100 2.24 β-D-AP 3 100 1.36 β-D-AQ 3 100 0.87 β-D-AT 4100 1.42 β-D-BE 3 100 1.23 β-D-BL 2 100 1.20 β-D-BO 3 100 0.80 β-D-BS 210 1.48 β-D-CL 6 40 3.10 β-D-CM 3 40 1.77 β-D-DJ 1 40 1.58 β-D-DK 2 1002.17 β-D-DL 2 100 1.33 β-D-HA 1 100 2.87 β-D-HB 2 100 2.26 β-D-MD 1 1002.16 β-D-ME 4 100 2.41 β-D-MF 4 100 1.41 β-D-QA 1 100 1.50 β-D-TA 1 1001.30 β-D-VA 1 100 4.69 β-L-FC 2 100 2.39

Example 56

[1530] Alternate Cell Culture Systems for Determining AntiviralActivities

[1531] The assay described above can be adapted to the other members ofthe Flaviviridae by changing the cell system and the viral pathogen.Methodologies to determine the efficacy of these antiviral compoundsinclude modifications of the standard techniques as described byHolbrook, M R et al. Virus Res. 2000, 69 (1), 31; Markland, W et al.Antimicrob. Agents. Chemother. 2000, 44 (4), 859; Diamond, M S et al.,J. Virol. 2000, 74 (17), 7814; Jordan, I. et al. J. Infect. Dis. 2000,182, 1214; Sreenivasan, V. et al. J. Virol. Methods 1993, 45 (1), 1; orBaginski, S G et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (14), 7981or the real-time RT-PCR technology. Specifically, an HCV replicon systemin HuH7 cells (Lohmann, V et al. Science, 1999, 285 (5424), 110) ormodifications thereof (Rice et al. 2000, abstract Xth InternationalSymposium for Viral Hepatitis and Liver Disease, Atlanta, Ga.) can beused.

Example 57

[1532] Cytotoxicity Testing of Candidate Compounds

[1533] The cytotoxicity testing as performed herein is a standardtechnique. Briefly, cells are seeded in 96-well plates at variousconcentrations (dependent on cell type, duration of assay), typically at5×10³ cells per well, in the presence of increasing concentrations ofthe test compound (0, 1, 3, 10, 33, and 100 μM). After three (Verocells), or four (CEM cells), or five (PBM cells) day-incubation, cellviability and mitochondrial activity are measured by adding the MTT-dye(Promega), followed by a 8 hours incubation. Afterwards the platescontaining the dye are fixed by adding a stop-solution followed byanother eight hour incubation. Finally, absorbance is read at 570 nm.Such methodologies are well described and available from themanufacturer (Promega).

[1534] A relevant list of compounds tested in this methodology is listedin Table 22. While the tested compounds are generally not cytotoxic,compound β-D-GA showed a selective cytotoxic effect on CEM cells. TABLE22 Cytotoxicity* of V-a and VIIIa ID PBM cells* CEM Cells* Vero Cells*β-D-GA >100 (11.3)    1.9 >57.4 β-D-GF >100 (−46.2)  >100 (11.2)   >100(4.3)    β-L-GA >100 (−113.2) >100 (1.1)    >100 (27.9)   β-L-GB >100(33)     >100 (8.3)    ˜171 β-L-GC >100 (−53.2)  >100 (−1.2)  >100(−13.4) β-L-GD >100 (−12.9)  >100 (−79.7) >100 (0.8)    β-L-GE >100(−59.7)  >100 (0.0)    >100 (10.6)   β-L-GF >100 (−70.4)  >100(35.1)   >100 (33.8)   β-L-GG >100 (−34.6)  >100 (17.3)   >100 (33.6)  β-L-GH >100 (−52.1)  >100 (19.7)   >100 (27.0)   β-L-GI >100(−47.8)  >100 (18.0)   >100 (31.9)  

Example 58

[1535] Antiviral Testing of Candidate Compounds for Respiratory Viruses

[1536] During the course of these experiments, compounds from generalformula (I) have ;.z 5 been tested for their antiviral activitiesagainst a set of viruses infecting the upper respiratory tract. Themethodologies used for these purposes are well described. The followingprotocols are standard operating procedures taken from the VirologyBranch, Division of Microbiology and Infectious Diseases, NIAID, NIH.

[1537] A. Viruses and Cell-Lines Used in Primary Screen

[1538] (i) Influenza A and B

[1539] Virus strains: A/Beijing/262/95 (HIN1) (Source CDC);A/Sydney/05/97 (H3N2) (source CDC); B/Beijing/184/93 (source: CDC).

[1540] Cell line: Maldin Darby Canine Kidney (MDCK)

[1541] (ii) Respiratory Syncytial Virus (RSV)

[1542] Virus strain A2 (source: ATCC).

[1543] Cell Line: African Green Monkey kidney (MA-104) cells

[1544] (iii) Parainfluenza Type 3 Virus

[1545] Virus Strain: 14702 (source: isolate 5/95 Boivin, MontrealCanada)

[1546] Cell line: African Green Monkey kidney (MA-104) cells

[1547] B. Methods for Antiviral Activity

[1548] (i) Inhibition of Viral Cytopathic Effect (CPE)

[1549] This test is run in 96-well micro-titer plates. In this CPEinhibition test, four loglo dilutions of each test compound will beadded to 3 cups containing the cell mono-layer; within 5 min, the virusis then added and the plate sealed, incubated at 37° C. and CPE readmicroscopically when untreated infected controls develop a 3 to 4+ CPE(approximately 72 to 120 hours). A known positive control drug isevaluated in parallel with test drug in each test. This drug isRibavirin for influenza, measles, RSV and para-influenza.

[1550] (ii) Increase in Neutral Red (NR) Dye Uptake.

[1551] This test is run to validate the CPE inhibition seen in theinitial test, and utilizes the same 96-well micro-plate after CPE hasbeen red. Neutral red is added to the medium; cells not damaged by virustake up greater amount of dye, which is read on a computerizedmicro-plate reader. The method as described by McManus (Appl.Environment. Microbiol. 31:35-38, 1976) is used. An EC₅₀ is determinedfrom this dye uptake.

[1552] (iii) Confirmatory Test: CPE-Visual and Virus Yield Assay

[1553] Compounds considered active by CPE inhibition and by NR dyeuptake will be retested using both CPE inhibition and effect onreduction of virus yield. Collected eluates from the initial testing areassayed for virus titer by serial dilution onto mono-layers ofsusceptible cells. Development of CPE in these cells is indicative forthe presence of infectious virus The EC₉₀, which is the drug thatinhibits the virus production by 1-log is determined from these data.

[1554] Table 23 summarizes the results of part of the antiviral testing.β-D-BS has potent anti-flaviviridae activity and potent in vitroantiviral capacities against influenza A and B, as well as someactivities against RSV. There is no activity against Parainfluenza type3 virus, illustrating that this compound is exerting a specificantiviral effect against certain classes of RNA viruses, but not all.

[1555] In addition, compound β-D-CL is a potent in-vitro anti-RSVcompound with a selectivity index of 150. TABLE 23 Antiviral effect onrespiratory viruses Initial Test, Antiviral Screening with RespiratoryViruses by CPE Inhibition (Visual) β-D-AJ β-D-BS β-D-CL β-D-DJ InfluenzaA EC₅₀(μM) 150 1.5 >5 >500 (H1N1) SI** 2 50 0 0 Influenza AEC₅₀(μM) >500 1.5 >5 >500 (H3N2) SI** 0 50 0 0 Influenza B EC₅₀(μM) 1500.5 >5 50 SI** 2 150 0 >10 RSV* EC₅₀(μM) >500 0.5 0.5 500 SI** 0 80 1500 Parainfluenza EC₅₀(μM) >500 >500 90 >500 Type 3 Virus SI** 0 0 0 0Initial Test, Antiviral Screening with Respiratory Viruses by NeutralRed β-D-AJ β-D-BS β-D-CL β-D-DJ Influenza A EC₅₀(μM) 150 1.2 8 >500(H1N1) SI** >3.3 116 1.1 0 Influenza A EC₅₀(μM) >500 4 >5 >500 (H3N2)SI** 0 20 0 0 Influenza B EC₅₀(μM) 150 1.2 >5 110 SI** >3.3 133 0 >4.5RSV* EC₅₀(μM) >500 <0.5 <0.5 >500 SI** 0 >30 >170 0 ParainfluenzaEC₅₀(μM) >500 40 40 500 Type 3 Virus SI** 0 1 1 >1 Confirmatory Test,Antiviral Screening with Respiratory Viruses by Visual (EC₅₀) β-D-BSInfluenza A EC₅₀(μM) 1.3 (H1N1) SI** >246 Influenza A EC₅₀(μM) 0.5(H3N2) SI** >640 Influenza B EC₅₀(μM) 0.6 SI** >533 Confirmatory Test,Antiviral Screening with Respiratory Viruses by Yield (EC₉₀) β-D-BSInfluenza A EC₅₀(μM) 0.4 (H1N1) SI** >800 Influenza A EC₅₀(μM) 0.32(H3N2) SI** >1000 Influenza B EC₅₀(μM) 0.6 SI** >533

Example 59

[1556] Antiviral Testing of Candidate Compounds for Flaviviridae

[1557] A. The HCV Replicon System in Huh7 Cells.

[1558] Huh7 cells harboring the HCV replicon can be cultivated in DMEMmedia (high glucose, no pyruvate) containing 10% fetal bovine serum, 1×non-essential Amino Acids, Pen-Strep-Glu (100 units/liter, 100microgram/liter, and 2.92 mg/liter, respectively) and 500 to 1000microgram/milliliter G418. Antiviral screening assays can be done in thesame media without G418 as follows: in order to keep cells inlogarithmic growth phase, seed cells in a 96-well plate at low density,for example 1000 cells per well. Add the test compound immediate afterseeding the cells and incubate for a period of 3 to 7 days at 37° C. inan incubator. Media is then removed, and the cells are prepared fortotal nucleic acid extraction (including replicon RNA and host RNA).Replicon RNA can then be amplified in a Q-RT-PCR protocol, andquantified accordingly. The observed differences in quantification ofreplicon RNA is one way to express the antiviral potency of the testcompound. A typical experiment demonstrates that in the negative controland in the non-active compounds-settings a comparable amount of repliconis produced. This can be concluded because the measured threshold-cyclefor HCV RT-PCR in both setting is close to each other. In suchexperiments, one way to express the antiviral effectiveness of acompound is to subtract the threshold RT-PCR cycle of the test compoundwith the average threshold RT-PCR cycle of the negative control. Thisvalue is called DeltaCt (ΔCt or DCt). A ΔCt of 3.3 equals a 1-logreduction (equals EC₉₀) in replicon production. Compounds that result ina reduction of HCV replicon RNA levels of greater than 2 ACt values (75%reduction of replicon RNA) are candidate compounds for antiviraltherapy. Such candidate compounds are belonging to structures withgeneral formula (I)-(XXIII). Table 24 gives the average ΔCt values(N=times tested) that can be obtained if the target compounds areincubated in the described way for 96 hours. As a positive control,recombinant interferon alfa-2a (Roferon-A, Hoffmann-Roche, New Jersey,USA) is taken alongside as positive control.

[1559] However, this HCV ΔCt value does not include any specificityparameter for the replicon encoded viral RNA-dependent RNA polymerase.In a typical setting, a compound might reduce both the host RNApolymerase activity and the replicon-encoded polymerase activity.Therefore, quantification of rRNA (or any other host RNA polymerase Iproduct) or beta-actin mRNA (or any other host RNA polymerase II) andcomparison with RNA levels of the no-drug control is a relativemeasurement of the effect of the test compound on host RNA polymerases.Table 24 also illustrates the ACt values for rRNA of the test compounds.

[1560] With the availability of both the HCV ΔCt data and the rRNA ΔCt,a specificity parameter can be introduced. This parameter is obtained bysubtracting both ΔCt values from each other. This results inDelta-DeltaCT values (ΔΔct or DDCt); a value above 0 means that there ismore inhibitory effect on the replicon encoded polymerase, a ΔΔct valuebelow 0 means that the host rRNA levels are more affected than thereplicon levels. The antiviral activity of tested compounds, expressedas ΔΔCt values, is given in Table 24. As a general rule, ΔΔCt valuesabove 2 are considered as significantly different from the no-drugtreatment control, and hence, exhibits appreciable antiviral activity.However, compounds with a ΔΔCt value of less than 2, but showing limitedmolecular cytotoxicty data (rRNA ΔCT between 0 and 2) are also possibleactive compounds.

[1561] In another typical setting, a compound might reduce the host RNApolymerase activity, but not the host DNA polymerase activity.Therefore, quantification of rDNA or beta-actin DNA (or any other hostDNA fragment) and comparison with DNA levels of the no-drug control is arelative measurement of the inhibitory effect of the test compound oncellular DNA polymerases. Table 25 illustrates the ΔCt values for rDNAof the test compounds.

[1562] With the availability of both the HCV ΔCt data and the rDNA ΔCt,a specificity parameter can be introduced. This parameter is obtained bysubtracting both ΔCt values from each other. This results in ΔΔCtvalues; a value above 0 means that there is more inhibitory effect onthe replicon encoded polymerase, a ΔΔCt value below 0 means that thehost rDNA levels are more affected than the replicon levels. Theantiviral activity of tested compounds, expressed as ΔΔCt values, isgiven in Table 25. As a general rule, ΔΔCt values above 2 are consideredas significantly different from the no-drug treatment control, andhence, is an interested compound for further evaluation. However,compounds with a ΔΔCt value of less than 2, but with limited molecularcytotoxicty (rDNA ACT between 0 and 2) may be desired.

[1563] Compounds that result in the specific reduction of HCV repliconRNA levels, but with limited reductions in cellular RNA and/or DNAlevels are candidate compounds for antiviral therapy. Candidatecompounds belonging to general formula group (I)-(XXIII) were evaluatedfor their specific capacity of reducing Flaviviridae RNA (including BVDVand HCV), and potent compounds were detected (Tables 21, 24 and 25).TABLE 24 Ave. HCV RNA Ave. rRNA ID n ΔCt ΔCt Ave. ΔΔCt β-D-AA 3 3.832.41 1.42 β-D-AI 3 2.93 2.43 0.48 β-D-AJ 22 2.92 1.74 1.18 β-D-AK 4 3.732.48 1.25 β-D-AL 2 3.08 2.72 0.36 β-D-AN 6 3.33 2.11 1.22 β-D-AO 1 4.102.13 1.97 β-D-AP 2 3.27 3.23 0.05 β-D-AQ 7 4.45 3.22 1.22 β-D-AT 2 3.713.07 0.64 β-D-BE 2 4.44 2.80 1.64 β-D-BF 2 4.37 2.69 1.68 β-D-BH 1 3.060.91 2.15 β-D-BJ 2 5.06 3.62 1.44 β-D-BL 1 2.28 1.93 0.35 β-D-BO 1 4.522.95 1.57 β-D-BS 40 4.89 1.05 3.83 β-D-BT 5 4.83 3.59 1.24 β-D-BU 4 3.462.18 1.06 β-D-BV 3 1.88 0.65 1.22 β-D-CC 6 5.04 4.82 0.21 β-D-DD 1 6.604.99 1.61 β-D-DH 3 4.13 2.91 1.21 β-D-DJ 5 3.51 3.62 −0.11 β-D-EB 1 3.331.42 1.90 β-D-FA 2 3.80 3.58 1.44 β-D-GA 4 6.04 2.10 3.93 β-D-HA 2 5.523.85 1.68 β-D-HB 5 2.94 1.65 1.30 β-D-KB 2 3.61 2.52 1.10 β-D-LA 3 3.854.10 0.89 β-D-MD 3 3.57 1.95 1.62 β-D-ME 1 2.89 1.25 1.64 β-D-MF 2 3.792.69 1.10 β-D-OE 1 4.51 4.20 0.31 β-D-QA 3 2.91 3.81 −0.89 β-D-RB 2 4.303.18 1.12 β-D-TA 1 4.00 3.31 0.69 β-D-UA 1 2.91 1.61 1.3 β-D-VA 1 5.564.17 1.39 β-L-FC 3 5.55 5.13 0.42 β-L-JB 1 3.65 4.55 −0.90 β-L-KA 1 4.104.84 −0.74 β-L-KC 2 1.19 1.35 −0.16 IFN 4 5.21 0.69 4.52 ribavirin 23.13 2.35 0.78

[1564] TABLE 25 Ave. HCV RNA Ave. rDNA ID N ΔCt ΔCt average ΔΔCt β-D-AA3 3.83 2.53 1.88 β-D-AI 1 3.76 −0.96 4.55 β-D-AJ 16 2.75 0.43 2.33β-D-AK 1 3.51 2.69 0.79 β-D-AL 1 3.18 2.56 0.61 β-D-AN 2 3.86 2.53 1.88β-D-AO 1 4.10 1.84 2.26 β-D-AP 2 3.27 2.26 1.02 β-D-AQ 3 4.75 1.78 2.73β-D-AT 1 3.81 2.43 1.43 β-D-BE 1 4.99 2.06 2.98 β-D-BF 1 5.27 2.04 3.28β-D-BH 1 3.06 1.42 1.64 β-D-BJ 1 4.34 0.81 3.53 β-D-BL 1 2.28 1.62 0.65β-D-BS 14 4.81 0.38 4.45 β-D-BT 2 4.44 1.17 3.39 β-D-BU 4 3.46 1.10 1.16β-D-BV 3 1.88 0.31 1.65 β-D-CC 3 5.84 2.17 3.66 β-D-DD 1 6.60 3.30 3.30β-D-DH 1 4.14 0.89 3.25 β-D-DJ 1 4.84 2.70 2.14 β-D-EB 1 3.33 0.96 2.37β-D-FA 2 3.80 1.92 0.78 β-L-FC 1 4.41 1.00 3.41 β-D-HA 1 5.12 2.04 3.16β-D-HB 1 1.90 1.19 0.40 β-D-KB 1 3.81 0.00 3.81 β-L-JB 1 3.65 1.20 2.45β-L-KA 1 4.10 0.42 3.69 β-L-KC 1 2.73 −0.81 3.54 β-D-LA 1 3.54 1.56 1.98β-D-MD 2 3.50 1.58 1.46 β-D-ME 1 2.89 1.53 1.36 β-D-MF 2 3.79 2.17 1.65β-D-OE 1 4.51 −0.04 4.60 β-D-QA 1 4.85 2.30 2.55 β-D-RB 1 4.00 1.27 2.74β-D-TA 1 4.00 3.07 0.93 β-D-UA 1 2.91 0.50 2.41

Example 60

[1565] Toxicity Profile of β-D-GA

[1566] Cytotoxicity testing as performed here are standard techniques.Briefly, cells are seeded in 96-well plates at various concentrations(dependent on cell type, duration of assay), typically at 5×10³ cellsper well, in the presence of increasing concentrations of the testcompound (0, 1, 3, 10, 33, and 100 μM). Depending on the cell-typeincubation with test compound can vary in time, but is usually withinthe range of 3 to 5 days. Cell viability and mitochondrial activity aremeasured by adding the MTT-dye (Promega), followed by eight hours ofincubation. Afterwards the plates containing the dye are fixed by addinga stop-solution followed by another eight hour incubation. Finally,absorbance is read at 570 nm. Such methodologies are well described andavailable from the manufacturer (Promega).

[1567] While the tested compounds are generally not cytotoxic,surprisingly enough β-D-GA showed a selective cytotoxic effect on CEMcells (Table 21). In order to explore the complete potential of thiscompound, a set of human malignant T and B cells and various tumor celllines were incubated with β-D-GA at varying concentrations, and afterthe absorbance was read, an IC₅₀ value was calculated. As a control,Ara-C, 5FU, and cycloheximide was taken alongside (Table 26).

[1568] β-D-GA has potent toxicity in human malignant T and B cells, butnot in human PBM cells and non-T or B neoplastic cells. Compared toAra-C and 5-FU, the anticancer activity of β-D-GA is highly selectivefor T and B cells. TABLE 26 Toxicity profile of β-D-GA against varioustumor cell lines (IC₅₀, μM)* β-D-GA Ara-C 5-FU Cycloheximide PBM >100 713.7 2.6 Vero >100 0.8 65 2.1 CEM 2.9 0.6 90.5 0.1 SUDHL-1 0.7 3.7 ∘ 0.3SupT1 0.3 ∘ 53.6 0.6 H9 1.4 ∘ 14.2 1 JY 3 ∘ 7.5 0.8 BL41 <1.0 ∘ 24.1 0.3LNCaP 45.7 ∘ 22.1 2.4 SK-MES-1 >100 ∘ 13.1 3.4 SK-MEL-28 >100 ∘ 11.2 1HEPG2 >100 ∘ 40.6 3.6 MCF-7 >100 ∘ 43.7 1.5

[1569] The prevention of β-D-GA-related cytotoxicity in CEM cells (humanT-cell lymphoma) and in the SUDHL-1 cells (human anaplastic large T-celllymphoma cell line) was studied by adding natural nucleosides. Thisexperiment was initiated by adding 50 μM of natural nucleosides into themedia, together with increasing concentration of β-D-GA. CEM cells wereseeded at 2500 cells per well and incubated for 4 days (=fast growingcell line with a doubling time of ˜1.3 days). SUDHL-1 cells were seededat 10,000 cells/well, and incubated for 3 days (=slow growing cell line,doubling time ˜3days). The result of this experiment is plotted in FIG.4. This figure illustrates that cytidine and uridine markedly preventβ-D-GA toxicity in SUDHL-1 cells and also in CEM cells (similar plot,not shown). 2′-Deoxycytidine has modest preventive activity effect.These data allow to conclude that β-D-GA is equally effective againstslower growing SUDHL-1 cells and fast growing CEM cells and thatCytidine and uridine prevent the compound related toxicity in both celllines. The action of β-D-GA may be related to synthesis and functions ofhost RNA molecules, but not DNA.

[1570] The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications will be obvious tothose skilled in the art from the foregoing detailed description of theinvention and may be made while remaining within the spirit and scope ofthe invention.

We claim:
 1. A method for the treatment or prophylaxis of hostexhibiting a Flaviviridae, Orthomyxoviridae or Paramyxoviridae viralinfection or abnormal cellular proliferation comprising administering aneffective amount of a compound of the general formula (I) or (II):

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D is hydrogen, alkyl, acyl, monophosphate, diphosphate,triphosphate, monophosphate ester, diphosphate ester, triphosphateester, phospholipid or amino acid; each W¹ and W² is independently CH orN; each X¹ and X² is independently hydrogen, halogen (F, Cl, Br or I),NH₂, NHR⁴, NR⁴R^(4′),NHOR⁴,NR⁴NR^(4′)R^(4″), OH, O⁴, SH or SR⁴; each Y¹is O, S or Se; each Z is CH₂ or NH; each R¹ and R^(1′) is independentlyhydrogen, lower alkyl, lower alkenyl, lower alkynyl, aryl, alkylaryl,halogen (F, Cl, Br or I), NH₂, NHR⁵, NR⁵R^(5′), NHOR⁵, NR⁵NHR^(5′),NR⁵NR^(5′)R^(5″), OH, OR⁵, SH, SR⁵, NO₂, NO, CH₂OH, CH₂R^(5′), CO₂H,CO₂R⁵, CONH₂, CONHR⁵, CON⁵R^(5′) or CN; each R² and R^(2′) independentlyis hydrogen or halogen (F, Cl, Br or I), OH, SH, OCH₃, SCH₃, NH₂, NHCH₃,CH═CH₂, CN, CH₂NH₂, CH₂O H, CO₂H. each R³ and R³ independently ishydrogen or halogen (F, Cl, Br or I), OH, SH, OCH₃, SCH₃, NH₂, NHCH₃,CH₃, C₂H₅, CH═CH₂, CN, CH₂NH₂, CH₂O H, CO₂H. each R⁴, R^(4′), R^(4″),R⁵, R^(5′) and R^(5″) independently is hydrogen, lower alkyl, loweralkenyl, aryl, or arylalkyl such as unsubstituted or substituted phenylor benzyl; such that for the nucleoside of the general formula (I) or(II) at least one of R² and R² is hydrogen and at least one of R³ andR^(3′) is hydrogen.
 2. The method of claim 1, wherein the β-D nucleosideof the formula (I-a) is selected from one of the following: X¹ Y¹ R¹R^(1′) R² R^(2′) R³ R^(3′) NH₂ O H H OH H H OH NH₂ O H H OH H H I NH₂ OH H OH H H Cl NH₂ O H H OH H H Br NH₂ O H H OH H H S—CN NH₂ O H H OH H HN₃ NH₂ O H H H Cl H OH NH₂ O H H H Br H OH NH₂ O H H H OH Br H NH₂ O H HH OH H H NH₂ O H H H OH O—Ms H NH₂ O H H H OH O—Ts H NH₂ O H H O—Ms H HOH NH₂ O H H Cl H H OH NH₂ O D D OH H H OH NH₂ O F H OH H H OH NH₂ O F HH OH H OH NH₂ O F H H OH H H NH₂ O F H H OH Cl H NH₂ O F H H OH Br H NH₂O F H H Cl H OH NH₂ O F H H OH O—Ts H NH₂ O F H H OH O—Ms H NH₂ O Cl H HOH O—Ms H NH₂ O Br H H OH O—Ms H NH₂ O Br H H OH O—Ts H NH₂ O Br H H OHCl H NH₂ O Br H H OH H OH NH₂ O Br H OH H H OH NH₂ O I H H OH O—Ms H NH₂O I H H OH Br H NH₂ O I H H OH O—Ts H NH₂ O I H H Cl H OH NH₂ O I H Br HH OH NH₂ O OH H OH H H OH NH₂ O NH₂ H H OH H OH NH₂ O CH₃ H H OH Cl HNH₂ NH H H OH H H OH NH₂ S H H H Se- H H phenyl NH-(2-Ph-Et) O H H OH HH OH NH—COCH₃ O H H OH H H OH NH—NH₂ O H H OH H H OH NH—NH₂ O F H OH H HOH NH—NH₂ O CH₃ H H OH H OH NH—OH O H H H OH H OH NH—OH O F H H OH H OHNH—OH O Br H H OH H OH NH—OH O I H H OH H OH NH—OH O H H OH H H OH OH OOH H OH H H OH OH O NH₂ H H OH H OH OH O F H OH H H OH OH O F H H O—Ts HOH OH O F H H O—Ms H O—Ms OH O F H H OH H OH OH O F H H OH H O—Ts OH O FH H H H OH O—Et O H H H O—Bz H O—Bz S—CH₃ O H H H F H OH SH O H H H OH HOH SH O F H H OH H OH N₃ O H H H H H H NH-(2-Ph-Et) O H H H OH H OH OH OOH H H OH H OH OH O H H H OH H H

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.3. The method of claim 1, wherein the β-D nucleoside of the formula(I-b) is selected from one of the following: X¹ X² W¹ R² R^(2′) R³R^(3′) OH NH₂ N H OH H OH OH NH₂ CH F H H OH NH-cyclohexyl H CH H H H HNH₂ H CH H OH H F NH₂ H CH H H H H NH₂ NH₂ N H OH H OH NH₂ NH₂ CH H OH HOH Cl H CH F H H H Cl I CH H O—Ac H O—Ac Cl H CH H OH H OH NH₂ H CH H OHH H Cl H CH H OH H H

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.4. The method of claim 1, wherein the β-D nucleoside of the formula(II-a) is selected from one of the following: X¹ Y¹ R¹ R^(1′) R² R³NH-Bz-(m-NO₂) O F H H H NH-Bz-(o-NO₂) O F H H H NH₂ O F H F H

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.5. The method of claim 1, wherein the β-D nucleoside of the formula(II-b) is selected from one of the following: X¹ X² W¹ R² R³ Cl H CH F HOH H CH H H NH₂ F CH H H NH₂ F CH F H NH₂ H CH H H OH NH₂ CH H H OH H CHH H

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.6. A method for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula (V) or (VII):

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, W¹, W², X¹, X², Y¹, Z, R¹, R^(1′), R^(1′), R², R³andR^(3′) is the same as defined previously; such that for the nucleosideof the general formula (V) or (VI), at least one of R² and R^(2′) ishydrogen and at least one of R³ and R^(3′) is hydrogen.
 7. The method ofclaim 6, wherein the β-D nucleoside of the formula (V-a) is selectedfrom one of the following: X¹ Y¹ R¹ R^(1′) R² R^(2′) R³ R^(3′) NH₂ O F HH OH H OH OH H CH₃ H H H H H OH O H H H H H H NH₂ O H H H OH H OH NH₂ OH H H H H H OH O F H H OH H OH NH₂ O I H H H H H NH₂ O I H H OH H OH NH₂O Cl H H OH H OH

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.8. The method of claim 6, wherein the β-D nucleoside of the formula(VII-a) is selected from one of the following: X¹ Y¹ R¹ R^(1′) R² R^(2′)R³ R^(3′) NH₂ O H H H OH H OH NH₂ O F H H OH H OH NH—OH O H H H OH H OH

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.9. The method of claim 6, wherein the β-D nucleoside of the formula(VII-b) is selected from the following: X¹ X² W¹ R² R^(2′) R³ R^(3′) NH₂H CH H OH H OH

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.10. A method for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula (XI):

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, W¹, W², X¹, X², Y¹, Z, R¹, R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously; each Z¹ and Z² independentlyis O, S, NR⁶ or Se; each R⁶ is hydrogen, lower alkyl or lower acyl. 11.The method of claim 10, wherein the 5-D nucleoside of the formula (XI-a)is selected from one of the following: X¹ Y¹ Z¹ Z² R¹ R¹ NH₂ O O O H HNH₂ O O S F H NH₂ O O O F H

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.12. The method of claim 10, wherein the E-D nucleoside of the formula(XI-b) is selected from one of the following: X¹ X² W¹ Z¹ Z² Cl H CH O SCl NH₂ CH O S NH₂ F CH O S OH H CH O O

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.13. A method for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula (XIII):

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, R¹, R^(1′), R², R^(2′), R³ and R^(3′) is the same asdefined previously; each Y² is O, S, NH or NR; each Y³ is O, S, NH orNR⁸; each X³ is OR⁹ or SR⁹; and each R⁷, R⁸ and R⁹ is hydrogen, loweralkyl of C₁-C₆, arylalkyl or aryl; such that for the nucleoside of thegeneral formula (XIII-d), at least one of R² and R^(2′) is hydrogen andat least one of R³ and R^(3′) is hydrogen.
 14. The method of claim 13,wherein the β-D nucleoside of the formula (XIII-a) is selected from oneof the following: Y² Y³ R¹ R^(1′) R² R^(2′) R³ R^(3′) O O F H H OH H OH

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.15. The method of claim 13, wherein the β-D nucleoside of the formula(XIII-c) is selected from one of the following: Y² Y³ R¹ R^(1′) R³R^(3′) O O F H H OH O O F H H O—Ms NH O H H H O—Ms NH O H H H O—Ac NH OH H H OH NH O F H H OH NH O F H H O—Ac

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.16. The method of claim 13, wherein the β-D nucleoside of the formula(XIII-d) is selected from the following: Y² X³ R¹ R^(1′) R² R^(2′) R³R^(3′) O O—CH₃ H H H O—Ac H O—Ac

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.17. A method for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula (XIV):

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, X¹, Y¹, Z¹, R¹, R², R^(2′), R³ and R^(3′) is the sameas defined previously; each L¹ is hydrogen, Cl or Br; each L² is OH,OCH₃, OC₂H₅, OC₃H₇, OCF₃, OAc or OBz; each Z³ can be O or CH₂.
 18. Themethod of claim 17, wherein the β-D nucleoside of the formula (XIV) isselected from one of the following: X¹ Y¹ R¹ R^(1′) R² R^(2′) R³ R^(3′)L¹ L² NH₂ O NH—OH OH OH H H OH H OH OH O O F H OH H OH Cl O—CH₃ OH O O HH OH H OH Br O—CH₃ OH O O F H OH H OH Br O—COCH₃ OH O O F H OH H OH BrO—CH₃ OH O O F H OH H OH Br O—Et OH O O Cl H OH H OH Br O—CH₃

or its β-L-enantiomer or its pharmaceutically acceptable salt thereof.19. A method for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula (XV):

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, W¹, W², X¹, Y¹, Z³, R¹, R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously.
 20. The method of claim 19,wherein the E-D nucleoside of the formula (XV-a) is defined as thefollowing: Y¹ Z³ R¹ R^(1′) R² R^(2′) R³ R^(3′) O O H H H OH H OH

its β-L-enantiomer or its pharmaceutically acceptable salt thereof. 21.The method of claim 19, wherein the β-D nucleoside of the formula (XV-b)is defined as the following: X¹ W¹ Z³ R² R^(2′) R³ R^(3′) NH₂ CH O H OHH OH

its β-L-enantiomer or its pharmaceutically acceptable salt thereof. 22.A method for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula (XVI):

or its β-L enantiomer or its pharmaceuticaily acceptable salt thereof,wherein: each D, W¹, X¹, X², Y¹, Z, R¹, R², R^(2′), R³ and R^(3′) is thesame as defined previously; each W³ is independently N, CH or CR¹; eachW⁴ and W⁵ is independently N, CH, CX¹ or CR¹; and each Z⁴and Z⁵ isindependently NH or C(═Y¹); such that if Z⁴ and Z⁵ are covalently bound,then Z⁴ is not C(═Y¹) when Z⁵ is C(═Y¹); and there are no more thanthree ring-nitrogens.
 23. The method of claim 22, wherein the β-Dnucleoside of the formula (XVI-a) is selected as one of the following:W³ Z⁴ W⁵ W⁴ Z⁵ R² R^(2′) R³ R^(3′) CH NCH₃ C—OH N C═O H OH H O—Ts CH NHC—NH₂ N C═O H OH H OH CH NH C—NHAc N C═O H OH H OH CH NH C—OH N C═O H OHH OH CH NCH₃ C—NH₂ N C═O H OH H OH CH NH C—NHBz N C═O H OH H OH CH C═OC—NH₂ C—SH NH H OH H OH CH NH C—OH N C═O H Cl H OH CH NH C—NH₂ N C═O HBr H OH

its β-L-enantiomer or its pharmaceutically acceptable salt thereof. 24.The method of claim 22, wherein the β-D nucleoside of the formula(XVI-c) is defined as the following: W³ Z⁴ Z⁵ W⁴ R² R^(2′) R³ R^(3′) CHN—CH₃ C═O N H OH H O—Ac

its β-L-enantiomer or its pharmaceutically acceptable salt thereof. 25.The method of claim 22, wherein the β-D nucleoside of the formula(XVI-d) is defined as the following: W³ Z⁴ Z⁵ W⁴ R³ R^(3′) CH N C═NH N HOH

its β-L-enantiomer or its pharmaceutically acceptable salt thereof. 26.The method of claim 22, wherein the β-D nucleoside of the formula(XVI-f) is defined as the following: X¹ X² W¹ R² R^(2′) R³ R^(3′) NH₂ HN H OH H OH

its β-L-enantiomer or its pharmaceutically acceptable salt thereof. 27.A method for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula (XVII):

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, W¹, W², X¹, X², Y¹, Z³, R¹, R^(1′), R², R²⁴ , R³ andR^(3′) is the same as defined previously; each X⁴ and X⁵ isindependently hydrogen, halogen (F, Cl, Br or I), N₃, NH₂, NHR⁸, NR⁸RROH, OR⁸, SH or SR; and each R⁸ and R^(8′) is independently hydrogen,lower alkyl, lower alkenyl, aryl or arylalkyl, such as an unsubstitutedor substituted phenyl or benzyl; such that for the nucleoside of thegeneral formula (XVII-a) or (XVII-b), X⁴ is not OH or OR⁸.
 28. Themethod of claim 27, wherein the β-D nucleoside of the formula (XVII-d)is defined as the following: X¹ X² W¹ X³ X⁴ NH₂ F CH H OH

its β-L-enantiomer or its pharmaceutically acceptable salt thereof. 29.A method for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula (XVIII):

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, W¹, W², X¹, X², Y¹, R¹ R^(1′), R², R^(2′), R³ andR^(3′) is the same as defined previously;
 30. A method for the treatmentor prophylaxis of host exhibiting a Flaviviridae, Orthomyxoviridae orParamyxoviridae viral infection or abnormal cellular proliferationcomprising administering an effective amount of a compound of thegeneral formula (XIX):

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, R¹, R⁴ and R^(4′) is the same as defined previously;each R⁹ is hydrogen, halogen (F, Cl, Br or I) or each P¹ is hydrogen,lower alkyl, lower alkenyl, aryl, arylalkyl (such as an unsubstituted orsubstituted phenyl or benzyl), OH, OR⁴, NH₂, NUR⁴ or NR⁴R^(4′); and eachP² and P³ is independently hydrogen, alkyl, acyl, -Ms, -Ts,monophosphate, diphosphate, triphosphate, mono-phosphate ester,diphosphate ester, triphosphate ester, phospholipid or amino acid.
 31. Amethod for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula:

or its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D and P² is the same as defined previously.
 32. A methodfor the treatment or prophylaxis of host exhibiting a Flaviviridae,Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellularproliferation comprising administering an effective amount of a compoundof the general formula (XX):

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P¹, P², P³, R¹, R⁴, R^(4′) and R⁹ is the same asdefined previously.
 33. A method for the treatment or prophylaxis ofhost exhibiting a Flaviviridae, Orthomyxoviridae or Paramyxoviridaeviral infection or abnormal cellular proliferation comprisingadministering an effective amount of a compound of the general formula(XXI):

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P¹, P², P³, R¹, R⁴ and R^(4′) is the same as definedpreviously.
 34. A method for the treatment or prophylaxis of hostexhibiting a Flaviviridae, Orthomyxoviridae or Paramyxoviridae viralinfection or abnormal cellular proliferation comprising administering aneffective amount of a compound of the general formula:

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P² and P³ is the same as defined previously.
 35. Amethod for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula (XXII):

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P¹ and R¹ is the same as defined previously.
 36. Amethod for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula:

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: D is the same as defined previously.
 37. A method for thetreatment or prophylaxis of host exhibiting a Flaviviridae,Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellularproliferation comprising administering an effective amount of a compoundof the general formula (XXIII):

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P¹, P², P³, R¹, R⁴ and R^(4′) is the same as definedpreviously.
 38. A method for the treatment or prophylaxis of hostexhibiting a Flaviviridae, Orthomyxoviridae or Paramyxoviridae viralinfection or abnormal cellular proliferation comprising administratingan effective amount of a compound of the general formula:

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P², and P³, is the same as defined previously.
 39. Amethod for the treatment or prophylaxis of host exhibiting aFlaviviridae, Orthornyxoviridae or Paramyxoviridae viral infection orabnormal cellular proliferation comprising administering an effectiveamount of a compound of the general formula:

or its pharmaceutically acceptable salt thereof.
 40. A method for thetreatment or prophylaxis of host exhibiting a Flaviviridae,Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellularproliferation comprising administering an effective amount of a compoundof the general formula:

or its pharmaceutically acceptable salt thereof.
 41. A method for thetreatment or prophylaxis of host exhibiting a Flaviviridae,Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellularproliferation comprising administering an effective amount of a compoundof the general formula:

or its pharmaceutically acceptable salt thereof.
 42. A method for thetreatment or prophylaxis of host exhibiting a Flaviviridae,Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellularproliferation comprising administering an effective amount of a compoundof the general formula (I) or (II):

or its pharmaceutically acceptable salt thereof.
 43. A method for thetreatment or prophylaxis of host exhibiting a Flaviviridae,Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellularproliferation comprising administering an effective amount of a compoundof the general formula:

or its pharmaceutically acceptable salt thereof.
 44. A method for thetreatment or prophylaxis of a hepatitis C virus infection in a hostcomprising administering an effective treatment amount of a compoundaccording to any one of claims 1-29.
 45. A method for the treatment orprophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a β-D nucleoside of theformula (XIX):

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, R¹, R⁴ and R^(4′) is the same as defined previously;each R⁹ is hydrogen, halogen (F, Cl, Br or I) or OP³; each P¹ ishydrogen, lower alkyl, lower alkenyl, aryl, arylalkyl (such as anunsubstituted or substituted phenyl or benzyl), OH, OR⁴, NH₂, NHR⁴ orNR⁴R^(4′); and each P² and P³ is independently hydrogen, alkyl, acyl,-Ms, -Ts, monophosphate, diphosphate, triphosphate, mono-phosphateester, diphosphate ester, triphosphate ester, phospholipid or aminoacid; optionally in a pharmaceutically acceptable carrier.
 46. A methodfor the treatment or prophylaxis of a hepatitis C virus infection in ahost comprising administering an effective treatment amount of a E-Dnucleoside of the formula:

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D and P² is the same as defined previously; optionally ina pharmaceutically acceptable carrier.
 47. A method for the treatment orprophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a 1-D nucleoside of theformula (XX):

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P¹, P², P³, R¹, R⁴, R^(4′) and R⁹ is the same asdefined previously; optionally in a pharmaceutically acceptable carrier.48. A method for the treatment or prophylaxis of a hepatitis C virusinfection in a host comprising administering an effective treatmentamount of a β-D nucleoside of the formula (XXI):

its 1-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P¹, P², P³, R¹, R⁴ and R^(4′) is the same as definedpreviously; optionally in a pharmaceutically acceptable camrer.
 49. Amethod for the treatment or prophylaxis of a hepatitis C virus infectionin a host comprising administering an effective treatment amount of aβ-D nucleoside of the formula:

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P² and P³ is the same as defined previously; optionallyin a pharmaceutically acceptable carrier.
 50. A method for the treatmentor prophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a β-D nucleoside of theformula (XXII):

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P¹ and R¹ is the same as defined previously; optionallyin a pharmaceutically acceptable carrier.
 51. A method for the treatmentor prophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a β-D nucleoside of theformula:

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: D is the same as defined previously; optionally in apharmaceutically acceptable carrier.
 52. A method for the treatment orprophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a β-D nucleoside of theformula (XXIII):

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P¹, P², P³, R¹, R⁴ and R^(4′) is the same as definedpreviously; optionally in a pharmaceutically acceptable carrier.
 53. Amethod for the treatment or prophylaxis of a hepatitis C virus infectionin a host comprising administering an effective treatment amount of aβ-D nucleoside of the formula (XXIII) is the following:

its β-L enantiomer or its pharmaceutically acceptable salt thereof,wherein: each D, P² and P³ is the same as defined previously; optionallyin a pharmaceutically acceptable carrier.
 54. A method for the treatmentor prophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a nucleoside of theformula:

or its pharmaceutically acceptable salt thereof; optionally in apharmaceutically acceptable carrier.
 55. A method for the treatment orprophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a nucleoside of theformula:

or its pharmaceutically acceptable salt thereof; optionally in apharmaceutically acceptable carrier.
 56. A method for the treatment orprophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a nucleoside of theformula:

or its pharmaceutically acceptable salt thereof; optionally in apharmaceutically acceptable carrier.
 57. A method for the treatment orprophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a nucleoside of theformula:

or its pharmaceutically acceptable salt thereof; optionally in apharmaceutically acceptable carrier.
 58. A method for the treatment orprophylaxis of a hepatitis C virus infection in a host comprisingadministering an effective treatment amount of a nucleoside of theformula:

or its pharmaceutically acceptable salt thereof; optionally in apharmaceutically acceptable carrier.